Inhibitors of mitochondrial fission and methods of use thereof

ABSTRACT

The present disclosure provides peptides and constructs that inhibit mitochondrial fission, and compositions comprising the peptides or constructs. The present disclosure provides methods of reducing abnormal mitochondrial fission in a cell. Also provided are methods for designing and validating mitochondrial fission inhibitor constructs and peptides, including but not limited to, evaluating the effects of the constructs and peptides on Drp1 GTPase activity, binding of Drp1 to Fis1, reduction of mitochondrial damage, reduction in cell death, inhibition of mitochondrial fragmentation in a cell under pathological conditions, and reduced loss of neurites in primary dopaminergic neurons in a Parkinsonism cell culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/471,221, filed May 14, 2012, now allowed, which claims the benefit ofU.S. Provisional Application No. 61/486,044, filed May 13, 2011, thecontents of which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. R01HL52141 awarded by the National Institutes of Health. The Government hascertain rights in this invention.

CROSS-REFERENCE TO A SEQUENCE LISTING

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created May 6, 2014, and named 0915118272US01seqlist.txt(57,842 bytes), the contents of which are incorporated herein byreference in their entirety.

BACKGROUND

Mitochondrial dysfunction plays an important role in a number of humandiseases, including neurodegenerative diseases, cardiovascular disease,diabetes, and cancer. Proper mitochondrial function is maintained, inpart, by balanced mitochondrial dynamics, i.e., a balance between anincrease in mitochondrial number by fission and a decrease inmitochondrial number by fusion.

Mitochondria are organized in a highly dynamic tubular network that iscontinuously reshaped by opposing processes of fusion and fission (Chan,2006, Annu Rev Cell Dev Biol, 22:79-99). This dynamic process controlsnot only mitochondrial morphology, but also the subcellular location andfunction of mitochondria. A defect in either fusion or fission limitsmitochondrial motility, decreases energy production and increasesoxidative stress, thereby promoting cell dysfunction and death(Jahani-Asl et al., 2010, Biochim Biophys Acta 1802:162-166; Scott andYoule, 2010; Essays Biochem, 47:85-98). The two opposing processes,fusion and fission, are controlled by evolutionarily conserved largeGTPases that belong to the dynamin family of proteins, In mammaliancells, mitochondrial fusion is regulated by mitofusin-1 and -2 (MFN-1/2)and optic atrophy 1 (OPA1), whereas mitochondrial fission is controlledby dynamin-1-related protein, Drp1 (Scott and Youle, 2010; EssaysBiochem, 47:85-98; Chan, 2006, Cell 125:1241-1252).

Drp1 is primarily found in the cytosol, but it translocates from thecytosol and the mitochondrial surface in response to various cellularstimuli to regulate mitochondrial morphology (Chang and Blackstone,2010; Ann NY Acad Sci, 1201:34-39). At the mitochondrial surface, Drp1is thought to wrap around the mitochondria to induce fission powered byits GTPase activity (Smirnova et al., 2001, Mol Biol Cell 12:2245-2256).Cell culture studies demonstrated that Drp1-induced excessivemitochondrial fission and fragmentation plays an active role inapoptosis (Frank et al., 2001, Dev Cell, 1:515-525; Estaquier andArnoult, 2007, Cell Death Differ, 14:1086-1094), autophagic cell death(Twig et al., 2008, EMBO J. 27:433-446; Barsoum et al., 2006, EMBO J.25:3900-3911) and necrosis (Wang et al., 2012, Cell 148:228-243).Inhibition of Drp1 by either expression of a Drp1-dominant negativemutant or by RNA interference leads to decreased mitochondrialfragmentation. This reduction in mitochondrial fission impairmentresults in longer and more interconnected mitochondrial tubules,increased ATP production, and the prevention of cell death (Frank etal., 2001, Dev Cell, 1:515-525; Barsoum et al., 2006, EMBO J.25:3900-3911; Yuan et al., 2007, Cell Death Differ, 14:462-471).

The association of Drp1 with the mitochondrial outer membrane and itsactivity in mammalian cells depends on various accessory proteins. Fis1is an integral mitochondrial outer membrane protein that recruits Drp1to promote fission (Yoon et al., 2003, Mol Cell Biol, 23:5409-5420;James et al., 2003, J Biol Chem 278:36373-36379). In yeast, recruitmentof Dnm1 (yeast Drp1) from the cytosol and assembly in punctatestructures on the mitochondrial surface depends on Fis1 (Fannjiang etal., 2004, Genes Dev 18:2785-2797; Suzuki et al., 2005, J Biol Chem280:21444-21452). In mammals, Fis1 interacts with Drp1 and apparentlyhas a similar role in mitochondrial fission as its yeast counterpart;Fis1 overexpression promotes mitochondrial fragmentation and Fis1depletion produces interconnected mitochondrial network (Yoon et al.,2003, Mol Cell Biol, 23:5409-5420; James et al., 2003, J Biol Chem278:36373-36379; 18).

Since protein-protein interaction (PPI) between Drp1 and Fis1 appear tobe required for mitochondrial fission, an inhibitor of this interactionmay have a therapeutic utility. A rational design protocol was used toidentify short peptide inhibitors of the protein-protein interactionbetween Drp1 and Fis1. Among other things, a novel selective peptideinhibitor of Drp1 was identified and its use as an inhibitor ofDrp1-mediated mitochondrial dysfunction in a cell culture model ofParkinson's disease (PD) was examined. Such mitochondria fissioninhibitor compositions are described herein as related to a need in theart for methods of reducing aberrant mitochondrial fission.

SUMMARY

The present disclosure provides peptides that inhibit mitochondrialfission, and compositions comprising the peptides. The presentdisclosure provides methods of reducing abnormal mitochondrial fissionin a cell as well as methods for treating diseases or disordersassociated with abnormal mitochondrial fission.

In one aspect, a mitochondrial fission inhibitor peptide is providedwherein the peptide comprises or consists of about 7 to 20 amino acids.In one embodiment, the peptide comprises an amino acid sequence havingat least about 80%, 85%, 90%, or 95% amino acid identity to a contiguousstretch of from about 7 to 20 amino acids of a Drp1 polypeptide or aFis1 polypeptide.

In one embodiment, the mitochondrial fission inhibitor peptide comprisesa peptide that is about 43%, 57%, 71%, or 86% identical to SEQ ID NO:12.In another embodiment, the fission inhibitor peptide comprises a peptidethat is about 89%, 78%, 67%, or 56% identical to SEQ ID NO:11. In stillanother embodiment, the fission inhibitory peptide comprises a peptidethat is about 89%, 78%, 67%, or 56% identical to SEQ ID NO:15. In oneembodiment, the fission inhibitor peptide comprises SEQ ID NO:12, SEQ IDNO:11 or SEQ ID NO:15.

In one aspect, a mitochondrial fission inhibitor construct is provided,wherein the construct comprises a mitochondrial fission inhibitorpeptide.

In one embodiment, the mitochondrial fission inhibitor construct furthercomprises a carrier moiety. In another embodiment, the carrier moiety isa carrier peptide. In still another embodiment, the carrier peptidecomprises SEQ ID NO:32.

In one embodiment, the mitochondrial fission inhibitor construct is alinear peptide which comprises the carrier peptide linked to the fissioninhibitor peptide by a peptide bond. In one embodiment, the fissioninhibitor construct further comprises a linker, wherein the linker ispositioned between the fission inhibitor peptide and the carrier peptideand the linker is linked at one end to the fission inhibitor peptide bya peptide bond and is linked at the other end to the carrier peptide bya peptide bond. In still another embodiment, the linker comprises 1, 2,3, 4, 5, or more amino acids. In another embodiment, the linkercomprises 1 to 2, 1 to 5, 2 to 5, 2 to 4, 1 to 10, 5 to 10, 3 to 6, or 2to 10 amino acids. In still another embodiment, the linker is G, GG,GGG, or GGGG (SEQ ID NO:62).

In one embodiment, the inhibitor construct comprises, in order fromamino terminus to carboxyl terminus: a) a protein transduction moiety,b) an optional linker, and c) a mitochondrial fission inhibitor peptide.

In one embodiment, the amino terminus, the carboxyl terminus or both theamino and carboxyl termini of the mitochondrial fission construct aremodified. In another embodiment, the amino terminal modification is anamine group or an acetyl group. In still another embodiment, thecarboxyl terminal modification is an amide group.

In one embodiment, the fission inhibitor construct comprises SEQ IDNO:18, SEQ ID NO: 19; SEQ ID NO:20, or SEQ ID NO:22.

In one embodiment, the fission inhibitor peptide, the carrier peptide,and/or the linker comprises one or more D-amino acids.

In one embodiment, the fission inhibitor construct inhibits GTPaseactivity of a Drp1 polypeptide. In another embodiment, the fissioninhibitor construct selectively inhibits GTPase activity of a Drp1polypeptide.

In one embodiment, the fission inhibitor construct inhibits binding of aFis1 polypeptide to a Drp1 polypeptide. In another embodiment, thefission inhibitor construct selectively inhibits binding of a Fis1polypeptide to a Drp1 polypeptide.

In one embodiment, the fission inhibitor construct reduces or inhibitsmitochondrial fragmentation in a cell. In another embodiment, thefission inhibitor construct reduces or inhibits fragmentation in a cellwhich has been stressed. In still another embodiment, the stress isoxidative stress or stress induced by MPP+, CCCP or rotenone.

In one embodiment, the Drp1 polypeptide is about 80%, 85%, 90%, 95%,98%, 99% or 100% identical to SEQ ID NO:1. In another embodiment, theFis1 polypeptide is about 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:2.

In another aspect, a pharmaceutical composition comprising amitochondrial fission inhibitor peptide is provided.

In one embodiment, the pharmaceutical composition comprises a carrierpeptide, an optional linker, and a mitochondrial fission inhibitorpeptide.

In one embodiment, the pharmaceutical composition comprises apharmaceutically acceptable excipient.

In one aspect, a method of inhibiting or reducing abnormal mitochondrialfission in a cell is provided, wherein the method comprises contactingthe cell with a composition comprising a mitochondrial fission inhibitorpeptide, wherein said contacting inhibits abnormal mitochondrialfission.

In one embodiment, the method comprises mixing the compositioncomprising a mitochondrial fission inhibitor peptide with a cell invitro. In another embodiment, the method comprises administering thecomposition to an animal, wherein the animal exhibits one or moresymptoms of a disease or disorder associated with abnormal mitochondrialfunction. In another embodiment, the administering to an animalcomprises administering an amount of the composition which is effectiveto reduce at least one adverse symptom of the disease. In still anotherembodiment, the adverse symptoms is selected from the group consistingof tremor, bradykinesia, rigidiy, and postural dysfunction.

In one aspect, a method of treating a disease or disorder associatedwith abnormal mitochondrial fission is provided.

In one embodiment, the method comprises administering to a subjectdiagnosed with the disease or disorder, or predisposed to the disease ordisorder, a therapeutically effective amount of a mitochondrial fissioninhibitor construct. In another embodiment, the administering iseffective to reduce at least one adverse symptom of the disease.

In one embodiment, the disease or disorder is Parkinson's disease,Huntington's disease, Alzheimer's disease, ischemia, reperfusion injury,diabetes-induced neuropathy or heart disease.

In one embodiment, the administering is by a route selected fromintravenous, intramuscular, subcutaneous or oral.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic showing regions of homology identified inthe Drp1 (SEQ ID NO:1) and Fis1 (SEQ ID NO:2) proteins.

FIG. 1B shows representative sequences identified in the homologousregions of Drp1 and Fis1 (DLLPRGT, SEQ ID NO:3; STQELLRFPK, SEQ ID NO:4;KSLAREQRD, SEQ ID NO:5; ELLPKGS, SEQ ID NO:6; SVEDLLKFEK, SEQ ID NO:7;KGSKEEQRD, SEQ ID NO:8).

FIG. 1C shows a sequence alignment of the 3 identified homology regionsof Drp1 (Regions 108, 109, 110) and Fis1 (Regions 111, 112, 113).

FIG. 2A is a graph showing effects of mitochondrial fission inhibitorpeptides on GTPase activity of Drp1.

FIG. 2B shows the effect of a mitochondrial fission inhibitor constructon GTPase activity of various proteins.

FIG. 2C shows the effects of mitochondrial fission inhibitor constructson interaction of Drp1 with mitochondria. The top panel is a westernblot; the bottom panel is a quantitative representation of the westernblot.

FIG. 2D shows the effects of mitochondrial fission inhibitor constructson interaction of Drp1 with Fis1. The top panel is a western blot; thebottom panel is a quantitative representation of the western blot.

FIG. 2E shows the effects of mitochondrial fission inhibitor constructson interaction of Drp1 with mitochondria. The top panel is a westernblot; the bottom panel is a quantitative representation of the westernblot.

FIG. 2F shows the effects of a mitochondrial fission inhibitor constructon Drp1 GTPase activity in the presence of MFN1 and OPA1.

FIG. 3A shows western blots in which mitochondria membrane preparationswere probed for the presence of proteins upon treatment with or withouta mitochondrial fission inhibitor construct, and in the presence orabsence of induced stress.

FIG. 3B shows a western blot in which mitochondria membrane preparationswere probed for the presence of proteins upon treatment with or withouta mitochondrial fission inhibitor construct, and in the presence orabsence of induced stress.

FIG. 3C shows a western blot in which mitochondria membrane preparationswere probed for the presence of proteins upon treatment with or withouta mitochondrial fission inhibitor construct, and in the presence orabsence of induced stress.

FIG. 3D shows a quantitative representation of Drp1 in mitochondrialmembranes upon treatment with or without a mitochondrial fissioninhibitor construct, and in the presence or absence of induced stress.

FIG. 3E shows a western blot in which mitochondria membrane preparationswere probed for the presence of proteins upon treatment with or withouta mitochondrial fission inhibitor construct, and in the presence orabsence of induced stress.

FIG. 3F shows a quantitative representation of phosphorylation of Drp1upon treatment with or without a mitochondrial fission inhibitorconstruct, and in the presence or absence of induced stress. Left panel:phosphorylation of wildtype Drp1; Right panel: phosphorylation of a Drp1having a Ser616 mutation.

FIG. 3G shows a quantitative representation of Drp1 in mitochondrialmembranes upon treatment with or without a mitochondrial fissioninhibitor construct, and in the presence or absence of induced stress.

FIG. 4A shows confocal microscopy photographs of mitochondria aftertreatment with mitochondrial fission inhibitor constructs.

FIG. 4B provides quantitative analysis of the confocal microscopyresults provided in FIG. 4A.

FIG. 5 provides quantitative data regarding mitochondrial fragmentationas affected by treatment with mitochondrial fission inhibitor constructsunder non-stressed and stressed conditions.

FIG. 6A provides quantitative data regarding superoxide production asaffected by treatment with mitochondrial fission inhibitor constructsunder stressed conditions.

FIG. 6B shows effects on mitochondrial membrane potential by treatmentwith mitochondrial fission inhibitor constructs under stressedconditions.

FIG. 6C shows effects on superoxide production by treatment withmitochondrial fission inhibitor constructs.

FIG. 6D shows a western blot indication effects of a mitochondrialinhibitor construct on cytochrome c release (top panel) and effects onmitochondrial membrane potential (bottom panel).

FIG. 6E shows effects of a mitochondrial inhibitor construct on totalreactive oxygen species (ROS) production under stressed conditions.

FIG. 7 shows effects of a mitochondrial inhibitor construct onmitochondrial superoxide production.

FIG. 8 shows the effects of a mitochondrial inhibitor construct onmitochondrial complex assembly under stressed conditions. Top panel:western blot in which probes detected complex I-V; bottom panel:quantitative data obtained from the western blot of the top panel.(*p<0.05 vs. MPP+-treated cells; **p<0.01 vs. MPP+-treated cells;#p<0.05 vs. control cells).

FIG. 9A shows a western blot showing the effects of a mitochondrialfission inhibitor construct on apoptosis marker levels under a stressedcondition.

FIG. 9B shows the effects of a mitochondrial fission inhibitor constructon levels of an apoptosis marker under a stressed condition.

FIG. 9C shows the effects of a mitochondrial fission inhibitor constructon levels of an autophagic marker under a stressed condition.

FIG. 9D shows the effects of a mitochondrial fission inhibitor constructon levels of an autophagic marker under a stressed condition. Leftpanel: western blot using a probe to detect LC3 I and LC3 II; rightpanel: quantitative data). (#p<0.05 vs. control group; *p<0.05, **p<0.01vs. MPP+-treated cells)

FIG. 9E shows the effects of mitochondrial fission inhibitor constructson cell viability under stressed conditions. (**p<0.01 vs. MPP+-treatedcells)

FIG. 9F shows the effects of mitochondrial fission inhibitor constructson cell survival under stressed conditions. (*, p<0.05; **, p<0.01 vs.control)

FIG. 10A shows effects of a mitochondrial fission inhibitor construct onmitochondrial superoxide production in a dopaminergic neuronal cellunder stressed conditions.

FIG. 10B shows effects of a mitochondrial fission inhibitor construct onmitochondrial fragmentation in a dopaminergic neuronal cell understressed conditions.

FIG. 10C shows effects of a mitochondrial fission inhibitor construct onneurite loss under stressed conditions.

DEFINITIONS

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include linear andcircular nucleic acids, messenger RNA (mRNA), cDNA, recombinantpolynucleotides, vectors, probes, and primers.

“Substantially pure” indicates that an entity (e.g., a synthetic peptideor a mitochondrial fission inhibitor peptide or construct) makes upgreater than about 50% of the total content of the composition (e.g.,total protein of the composition), or greater than about 80% of thetotal protein content. For example, a “substantially pure” refers tocompositions in which at least 80%, at least 85%, at least 90% or moreof the total composition is the entity of interest (e.g. 95%, 98%, 99%,greater than 99%), of the total protein. The protein can make up greaterthan about 90%, or greater than about 95% of the total protein in thecomposition.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a member or members of any mammalianor non-mammalian species that may have a need for the pharmaceuticalmethods, compositions and treatments described herein. Subjects andpatients thus include, without limitation, primate (including humans andnon-human primates), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. In some cases, thesubject is a murine (e.g., rat or mouse), such as a rat or mouse modelof a disease. In some cases, the subject is a human.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient, in combination withanother agent, or alone (e.g., in monotherapy) in one or more doses, toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and its severityand the age, weight, etc., of the subject to be treated.

The terms “mitochondrial fission inhibitor peptide,” “mitochondrialinhibitory peptide,” “mitochondrial inhibiting peptide,” or “subjectsynthetic peptide” are used interchangeably herein to refer to thepeptides described herein which function to inhibit mitochondrialfission as well as to inhibit one or more functions associated withmitochondrial fission as described herein.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amitochondrial fission inhibitor” peptide or construct includes aplurality of such peptides or constructs and reference to “the proteintranslocation domain” or “the protein translocation moiety” includesreference to one or more protein translocation domains and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides synthetic peptides that inhibitmitochondrial fission, and compositions comprising the peptides. Thepresent disclosure provides methods of reducing abnormal mitochondrialfission in a cell.

Excessive mitochondrial fragmentation through a process called fissionhas been implicated in the pathogenesis of diverse human diseases,including neurodegenerative diseases. Thus, selective inhibitors ofaberrant mitochondrial fission will provide important research tools aswell as potential leads for drug development. Disclosed herein aremethods for identifying inhibitors of protein-protein interaction (PPI)between the fission protein, Drp1 (Dynamin 1-like protein, GenBank Acc.No. AAH24590; SEQ ID NO:1) or an isoform thereof (e.g., GenBank Acc. No.000429; SEQ ID NO:9), and its mitochondrial adaptor, Fis1 (mitochondrialfission 1 protein, GenBank Acc. No. NP_(—)057152; SEQ ID NO:2) or anisoform thereof. Also disclosed are mitochondrial fission inhibitorpeptides identified through these methods.

Mitochondrial Fission Inhibitory Compositions

Previous studies have shown that short peptides derived from interactionsites between two proteins act as highly specific inhibitors of thatinteraction and are effective drugs in basic research and in animalmodels of human diseases, such as myocardial infarction and hypertension(Inagaki et al., 2003, Circulation 108:2304-2307; Qi et al., 2008, JClin Invest, 118:173-182; Palaniyandi et al., 2009, Cardiovasc Res82:229-239). One possibility is that because such peptides are flexibleand represent part of the natural binding site, they may be superior andmore selective inhibitors of protein-protein interaction as comparedwith more rigid small molecules (Rob and Mochly-Rosen, 1995, Proc NatlAcad Sci USA 92:492-496; Qvit and Mochly-Rosen, 2010, Drug Discov TodayDis Mech 7:e87-e93; Souroujon and Mochly-Rosen, 1998, Nat Biotechnol16:919-924). For example, a peptide corresponding to a homologoussequence between protein kinase C (PKC) and its scaffold protein, RACK,serves as a selective regulator of the function of PKC, as determined inculture and in in vivo animal models of acute myocardial infarction(Chen et al., 2001, Proc Natl Acad Sci USA 98:11114-11119; Kheifets etal., 2006, J Biol Chem 281:23218-23226; Dorn et al., 1999, Proc NatlAcad Sci USA, 96:12798-128803), heart failure (Inagaki et al., 2008,Hypertension 51:1565-1569), pain (Sweitzer et al., 2004, Pain110:281-289), and cancer (Kim et al., 2011, Prostate 71:946-954).

Using this rational approach, novel and selective peptide inhibitors ofexcessive mitochondrial fission can be designed and validated. Suchfission inhibitor peptides selectively inhibit the GTPase activation ofthe mitochondrial fission protein, Drp1.

L-ALIGN sequence alignment software (Huang, 1991, Advances in AppliedMathematics 12:337-357) used to align Drp1 and Fis1 identified 3different regions of sequence similarity between the two proteins (FIG.1A). The amino acid sequence for each of the 3 regions within each ofthe Drp1 and Fis1 proteins are presented in FIG. 1B. Based on empiricalstructure data and molecular modeling, it was determined that eachregion is present on the surface of Drp1 or Fis1, and thus likelyaccessible for protein-protein interaction between the two proteins.Further, using principles similar to the evolutionary trace method ofLichtarge and collaborators (Lichtarge et al., 1996, J Mol Biol257:342-358), it was found that while these homologous sequences areconserved in a variety of species, only the sequence in region 110 isidentical in mammals, fish, chicken and yeast, suggesting that thisregion is most likely critical for the function of Drp1 (FIG. 1C).Another way to determine whether region 110 in Drp1 may represent aunique site for protein-protein interaction is to determine whether itis present in other proteins in the human genome. Sixteen other proteinshave a sequence that is at least 80% similar to the sequence in region110. Such region-1,0-like sequences were found in TOM22 (mitochondrialimport receptor subunit, TOM22), DYN1 (dynamin-1), DYN2 (dynamin-2),DYN3 (dynamin-3), MIA3 (melanoma inhibitory activity protein 3), SCN5A(sodium channel protein type 5, subunit alpha), HIP1(Huntingtin-interacting protein 1), PCDGK (protocadherin gamma-C3),BI2L2 (brain-specific angiogenesis inhibitor 1-associated protein 2-likeprotein 2), ZSWIM5 (zinc finger, SWIM-type containing 5). ADAM 17(disintegrin and metalloproteinase domain-containing protein 17), AP2B(AP-2 complex subunit beta), ZSWM4 (zinc finger SWIM domain-containingprotein 4), MIA2 (melanoma inhibitory activity protein 3), CYP2W1(cytochrome P450, 2W1) and MSLN (mesothelin). However, Fis1 was the onlyprotein in which this sequence was 100% identical in other mammalians(and 50% identical in yeast), further supporting the hypothesis that 110represents an important region for interaction between Drp1 and Fis1.

A mitochondrial fission inhibitor construct or peptide inhibitsmitochondrial fission in a cell under pathological conditions, but doesnot inhibit mitochondrial fission in normal control cells. Thus, amitochondrial fission inhibitor construct or peptide of the presentdisclosure is useful for inhibiting aberrant (pathological)mitochondrial fission.

A mitochondrial fission inhibitor peptide can have a length of fromabout 7 amino acids to about 50 amino acids, e.g., from about 7 aminoacids to about 10 amino acids, from about 10 amino acids to about 15amino acids, from about 15 amino acids to about 20 amino acids, fromabout 20 amino acids to about 25 amino acids, from about 25 amino acidsto about 30 amino acids, from about 30 amino acids to about 35 aminoacids, from about 35 amino acids to about 40 amino acids, from about 40amino acids to about 45 amino acids, or from about 45 amino acids toabout 50 amino acids, or longer than 50 amino acids.

A mitochondrial fission inhibiting peptide can have a length of fromabout 7 amino acids to about 20 amino acids, e.g., a mitochondrialfission inhibiting peptide can have a length of 7 amino acids (aa), 8aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa.

In some cases, a mitochondrial fission inhibitor construct comprises, inorder from NH₂ (amino) terminus to COOH (carboxyl) terminus: a) acarrier peptide; b) an optional linker of from about 1 amino acid toabout 40 amino acids; and c) a mitochondrial fission inhibitor peptide.

In some cases, a mitochondrial fission inhibitor peptide comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 7 amino acids to about 20 amino acids of the Fis1 amino acidsequence (SEQ ID NO:2). A mitochondrial fission inhibitor peptide cancomprise an amino acid sequence differing in amino acid sequence by one,two, three, four, or five amino acids, compared to a contiguous stretchof from about 7 amino acids to about 20 amino acids of the Fis1 aminoacid sequence (SEQ ID NO:2). The amino acid differences can beconservative amino acid differences.

In some cases, a mitochondrial fission inhibitor peptide comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 7 amino acids to about 20 amino acids of the Drp1 amino acidsequence (SEQ ID NO:1). A mitochondrial fission inhibitor peptide cancomprise an amino acid sequence differing in amino acid sequence by one,two, three, four, or five amino acids, compared to a contiguous stretchof from about 7 amino acids to about 20 amino acids of the Drp1 aminoacid sequence (SEQ ID NO:1). The amino acid differences can beconservative amino acid differences.

By “conservative amino acid substitution” generally refers tosubstitution of amino acid residues within the following groups:

-   -   1) L, I, M, V, F;    -   2) R, K;    -   3) F, Y, H, W, R;    -   4) G, A, T, S;    -   5) Q, N; and    -   6) D, E.

Conservative amino acid substitutions in the context of a mitochondrialfission inhibitor peptide are selected so as to preserve activity of thepeptide. Such presentation may be preserved by substituting with anamino acid with a side chain of similar acidity, basicity, charge,polarity, or size to the side chain of the amino acid being replaced.Guidance for substitutions, insertion, or deletion may be based onalignments of amino acid sequences of different variant proteins orproteins from different species. For example, at certain residuepositions that are fully conserved, substitution, deletion or insertionmay not be allowed while at other positions where one or more residuesare not conserved, an amino acid change can be tolerated. Residues thatare semi-conserved may tolerate changes that preserve charge, polarity,and/or size. For example, a mitochondrial fission inhibitor peptidecomprising SEQ ID NO:12 may have 1, 2 or 3 amino acid substitutions, atposition 1, 2, 3, 4, 5, 6, and/or 7, wherein the substituted amino acidmay be any one of the known 20 amino acids, wherein the inhibitorpeptide maintains a mitochondrial fission inhibiting function.

A Fis1 polypeptide can have at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 100 amino acids to about 120 amino acids, from about 120amino acids to about 130 amino acids, from about 130 amino acids toabout 140 amino acids, or from about 140 amino acids to 152 amino acids,of the amino acid sequence depicted by SEQ ID NO:1.

A Drp1 polypeptide can have at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 500 amino acids to about 600 amino acids, or from about 600amino acids to about 650 amino acids, from about 650 amino acids toabout 675 amino acids, or from about 675 amino acids to 710 amino acids,of the amino acid sequence depicted by SEQ ID NO:2.

Protein Transduction Moiety

As noted above, a mitochondrial fission inhibitor construct can include,in addition to a mitochondrial fission inhibitor peptide, a carriermoiety (also referred to herein as a “protein transduction moiety”).“Carrier moiety” refers to a polypeptide, polynucleotide, carbohydrate,or organic or inorganic compound that facilitates traversing a lipidbilayer, micelle, cell membrane, organelle membrane, or vesiclemembrane. A carrier moiety attached to another molecule facilitates themolecule traversing a membrane, for example going from extracellularspace to intracellular space, or cytosol to within an organelle. In somecases, a carrier moiety facilitates crossing the blood-brain barrier. Insome embodiments, a carrier moiety is covalently linked to the aminoterminus of a mitochondrial fission inhibiting peptide. In someembodiments, a carrier moiety is covalently linked to the carboxylterminus of a mitochondrial fission inhibiting peptide.

In some cases, the carrier moiety is a carrier peptide and is covalentlylinked to a fission inhibiting peptide. In some embodiments, thecovalent linkage is a peptide bond. For example, the carrier peptide canbe a polypeptide having a length of from about 5 amino acids (aa) toabout 50 aa, e.g., from about 5 as to about 10 aa, from about 10 as toabout 15 aa, from about 15 as to about 20 aa, from about 20 as to about25 aa, from about 25 as to about 30 aa, from about 30 as to about 40 aa,or from about 40 as to about 50 aa.

Exemplary protein transduction domains which may be linked to themitochondria fission inhibitor peptide include but are not limited to aminimal undecapeptide protein transduction domain corresponding toresidues 47-57 of human immunodeficiency virus-1 (HIV-1) TAT (GenBankAcc. No. AEB53027; including YGRKKRRQRRR (SEQ ID NO:31;) or RRRQRRKKRGY(SEQ ID NO:32)), a polyarginine sequence comprising a number ofarginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7,8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002)Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia proteintransduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); atruncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:33); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:34);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:35); and RQIKIWFQNRRMKWKK(SEQ ID NO:36). Exemplary PTDs include but are not limited to,YGRKKRRQRRR (SEQ ID NO:37); RRRQRRKKRGY (SEQ ID NO:38); RKKRRQRRR (SEQID NO:39); an arginine homopolymer of from 3 arginine residues to 50arginine residues; Exemplary PTD domain amino acid sequences include,but are not limited to, any of the following: YGRKKRRQRRR (SEQ IDNO:40); RRRQRRKKRGY (SEQ ID NO:41); RKKRRQRR (SEQ ID NO:42); YARAAARQARA(SEQ ID NO:43); THRLPRRRRRR (SEQ ID NO:44); and GGRRARRRRRR (SEQ IDNO:45).

Linkers

Where a mitochondrial fission inhibitor construct includes a linkerwhich joins or links a carrier moiety to a mitochondrial fissioninhibitor peptide, the linker may be a peptide having any of a varietyof amino acid sequences. A linker which is a spacer peptide, can be of aflexible nature, although other chemical linkages are not excluded. Alinker peptide can have a length of from about 1 amino acid to about 40amino acids, e.g., from about 1 amino acid (aa) to about 5 aa, fromabout 5 as to about 10 aa, from about 10 as to about 20 aa, from about20 as to about 30 aa, or from about 30 as to about 40, in length. Theselinkers can be produced using synthetic, linker-encodingoligonucleotides to couple the proteins. Peptide linkers with a degreeof flexibility can be used. The linking peptides may have virtually anyamino acid sequence, where in some embodiments the linker peptide willhave a sequence that results in a generally flexible peptide. The use ofsmall amino acids, such as glycine and alanine, are of use in creating aflexible peptide. The creation of such sequences is routine to those ofskill in the art. Various linkers are commercially available and areconsidered suitable for use.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 40 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linker which can be used to join or link a carriermoiety to a mitochondrial fission inhibitor peptide, for example, viapeptide bonds, include glycine polymers (G)_(n), (e.g., where n is aninteger from 1 to about 20); glycine-serine polymers (including, forexample, (GS)_(n), GSGGS_(n) (SEQ ID NO:46) and GGGS_(n) (SEQ ID NO:47),where n is an integer of at least one), glycine-alanine polymers,alanine-serine polymers, and other flexible linkers known in the art.Glycine and glycine-serine polymers are of interest since both of theseamino acids are relatively unstructured, and therefore may serve as aneutral tether between components. Glycine polymers are used in someembodiments. See Scheraga, Rev. Computational Chem. 11173-142 (1992).Exemplary flexible linkers include, but are not limited to GG, GGG, GGS,GGSG (SEQ ID NO:48), GGSGG (SEQ ID NO:49), GSGSG (SEQ ID NO:50), GSGGG(SEQ ID NO:51), GGGSG (SEQ ID NO:52), GSSSG (SEQ ID NO:53), and thelike.

Non-peptide linker moieties can also be used to join or link a carriermoiety to a mitochondrial fission inhibitor peptide. The linkermolecules are generally about 6-50 atoms long. The linker molecules mayalso be, for example, aryl acetylene, ethylene glycol oligomerscontaining 2-10 monomer units, diamines, diacids, amino acids, orcombinations thereof. Other linker molecules which can bind topolypeptides may be used in light of this disclosure.

In an alternative embodiment, the inhibitor peptide may be linked to thecarrier peptide by a disulfide bond. In some embodiments, the disulfidebond is formed between two cysteines, two cysteine analogs or a cysteineand a cysteine analog. In this embodiment, both the modulatory peptideand the carrier peptide contain at least one cysteine or cysteineanalog. The cysteine residue or analog may be present as the N-terminalor C-terminal residue of the peptide or as an internal residue of theinhibitor peptide and of the carrier peptide. The disulfide linkage isthen formed between the sulfur residues on each of the cysteine residuesor analogs. Thus, the disulfide linkage may form between, for example,the N-terminus of the inhibitor peptide and the N-terminus of thecarrier peptide, the C-terminus of the inhibitor peptide and theC-terminus of the carrier peptide, the N-terminus of the inhibitorpeptide and the C-terminus of the carrier peptide, the C-terminus of theinhibitor peptide and the N-terminus of the carrier peptide, or anyother such combination including at any internal position within theinhibitor peptide and/or the carrier peptide.

Exemplary Peptides

Non-limiting examples of mitochondrial fission inhibitor peptidesinclude, e.g.:

STQELLRFPK; (SEQ ID NO: 10) KLSAREQRD; (SEQ ID NO: 11) DLLPRGS;(SEQ ID NO: 12) DLLPRGT; (SEQ ID NO: 13) CSVEDLLKFEK; (SEQ ID NO: 14)KGSKEEQRD; (SEQ ID NO: 15) and ELLPKGS. (SEQ ID NO: 16)

Each of these inhibitor peptides can be included in a mitochondrialfission inhibitor construct. A mitochondrial fission inhibiting peptidecan comprise an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, amino acid sequence identity to any of theabove-listed amino acid sequences. A mitochondrial fission inhibitingpeptide can comprise an amino acid sequence differing in amino acidsequence by one, two, three, four, or five amino acids, compared to anyof the above-listed amino acid sequences:

Non-limiting examples of a mitochondrial fission inhibitor constructinclude the following:

RRRQRRKKRGYGGSTQELLRFPK; (SEQ ID NO: 17) RRRQRRKKRGYGGKLSAREQRD;(SEQ ID NO: 18) RRRQRRKKRGYGGDLLPRGS; (SEQ ID NO: 19)RRRQRRKKRGYGGDLLPRGT; (SEQ ID NO: 20) RRRQRRKKRGYGGCSVEDLLKFEK;(SEQ ID NO: 21) RRRQRRKKRGYGGKGSKEEQRD; (SEQ ID NO: 22) andRRRQRRKKRGYGGELLPKGS. (SEQ ID NO: 23)

A mitochondrial fission inhibitor peptide can comprise an amino acidsequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, aminoacid sequence identity to any of the above-listed amino acid sequences.A mitochondrial fission inhibitor peptide can comprise an amino acidsequence differing in amino acid sequence by one, two, three, four, orfive amino acids, compared to any of the above-listed amino acidsequences.

Modifications

In some cases, a subject peptide comprises one or more modifications.For example, a mitochondrial fission inhibitor construct or peptide canbe cyclized. As another example, a subject peptide can have one or moreamino acid modifications. A subject mitochondrial fission inhibitorconstruct or peptide can include one or more D-amino acids.

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced arepeptides that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Also provided in the subject disclosure are mitochondrial fissioninhibitor constructs or peptides that have been modified using ordinarymolecular biological techniques and synthetic chemistry so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such peptides include those containing residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring synthetic amino acids.

A subject mitochondrial fission inhibitor construct or peptide may bejoined to a wide variety of other oligopeptides or proteins for avariety of purposes. By providing for expression of the subjectpeptides, various post-translational modifications may be achieved. Forexample, by employing the appropriate coding sequences, one may providefarnesylation or prenylation. For example, a mitochondrial fissioninhibitor construct or peptide can be bound to a lipid group at aterminus, so as to be able to be bound to a lipid membrane, such as aliposome.

Other suitable modifications include, but are not limited to (1)end-cappings of the terminal of the peptides, such as amidation of theC-terminus and/or acetylation or deamination of the N-terminus; (2)introducing peptidomimetic elements in the structure; and (3)cyclization, in which the cyclization of the peptide can occur throughnatural amino acids or non-naturally-occurring building blocks.

A subject mitochondrial fission inhibitor construct or peptide can be apeptoid (N-substituted oligoglycines), e.g., in which an amino acid sidechain is connected to the nitrogen of the peptide backbone, instead ofthe α-carbon. See, e.g., Zuckermann et al. (1992) J. Am. Chem. Soc.114:10646.

A subject mitochondrial fission inhibitor construct or peptide caninclude naturally-occurring and non-naturally occurring amino acids. Asubject mitochondrial fission inhibitor construct or peptide cancomprise D-amino acids, a combination of D- and L-amino acids, andvarious “designer” amino acids (e.g., β-methyl amino acids, Cα-methylamino acids, and Nα-methyl amino acids, etc.) to convey specialproperties to peptides.

Additionally, a subject mitochondrial fission inhibitor construct orpeptide can be a cyclic peptide. A subject mitochondrial fissioninhibitor construct or peptide can include non-classical amino acids inorder to introduce particular conformational motifs. Any knownnon-classical amino acid can be used. Non-classical amino acids include,but are not limited to, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate;(2S,3S)-methylphenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine;2-aminotetrahydronaphthalene-2-carboxylic acid;hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; β-carboline (D andL); HIC (histidine isoquinoline carboxylic acid); and HIC (histidinecyclic urea). Amino acid analogs and peptidomimetics can be incorporatedinto a subject mitochondrial fission inhibitor construct or peptide toinduce or favor specific secondary structures, including, but notlimited to, LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), aβ-turn inducing dipeptide analog; β-sheet inducing analogs; β-turninducing analogs; α-helix inducing analogs; γ-turn inducing analogs;Gly-Ala turn analog; amide bond isostere; tretrazol; and the like.

A subject mitochondrial fission inhibitor construct or peptide can be adepsipeptide, e.g., a linear or a cyclic depsipeptide. Kuisle et al.(1999) Tet. Letters 40:1203-1206. “Depsipeptides” are compoundscontaining a sequence of at least two alpha-amino acids and at least onealpha-hydroxy carboxylic acid, which are bound through at least onenormal peptide link and ester links, derived from the hydroxy carboxylicacids, where “linear depsipeptides” may comprise rings formed throughS—S bridges, or through an hydroxy or a mercapto group of an hydroxy-,or mercapto-amino acid and the carboxyl group of another amino- orhydroxy-acid but do not comprise rings formed only through peptide orester links derived from hydroxy carboxylic acids. “Cyclicdepsipeptides” are peptides containing at least one ring formed onlythrough peptide or ester links, derived from hydroxy carboxylic acids.

A subject mitochondrial fission inhibitor construct or peptide can becyclic or bicyclic. For example, the C-terminal carboxyl group or aC-terminal ester can be induced to cyclize by internal displacement ofthe —OH or the ester (—OR) of the carboxyl group or ester respectivelywith the N-terminal amino group to form a cyclic peptide. For example,after synthesis and cleavage to give the peptide acid, the free acid isconverted to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Methods for making cyclic peptides are well known in the art.See, e.g., U.S. Patent Publication No. 2011/0092384.

The term “bicyclic” refers to a peptide comprising two ring closures.The ring closures are formed by covalent linkages between amino acids inthe peptide. A covalent linkage between two nonadjacent amino acidsconstitutes a ring closure, as does a second covalent linkage between apair of adjacent amino acids which are already linked by a covalentpeptide linkage. The covalent linkages forming the ring closures may beamide linkages, i.e., the linkage formed between a free amino on oneamino acid and a free carboxyl of a second amino acid, or linkagesformed between the side chains or “R” groups of amino acids in thepeptides. Thus, bicyclic peptides may be “true” bicyclic peptides, i.e.,peptides cyclized by the formation of a peptide bond between theN-terminus and the C-terminus of the peptide, or they may be“depsi-bicyclic” peptides, i.e., peptides in which the terminal aminoacids are covalently linked through their side chain moieties.

A desamino or descarboxy residue can be incorporated at a terminus orterminii of the peptide, so that there is no terminal amino or carboxylgroup, to decrease susceptibility to proteases or to restrict theconformation of the peptide. C-terminal functional groups include amide,amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, andcarboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

In some embodiments, a subject mitochondrial fission inhibitor constructor peptide comprises one or more non-naturally occurring amino acids(e.g., non-encoded amino acids). In some embodiments, the non-naturallyencoded amino acid comprises a carbonyl group, an acetyl group, anaminooxy group, a hydrazine group, a hydrazide group, a semicarbazidegroup, an azide group, or an alkyne group. See, e.g., U.S. Pat. No.7,632,924 for suitable non-naturally occurring amino acids.

Inclusion of a non-naturally occurring amino acid can provide forlinkage to a polymer, a second polypeptide, a scaffold, etc. Forexample, a subject mitochondrial fission inhibitor construct or peptidelinked to a water-soluble polymer can be made by reacting awater-soluble polymer (e.g., poly(ethylene glycol) (PEG)) that comprisesa carbonyl group to an the subject mitochondrial fission inhibitorconstruct or peptide that comprises a non-naturally encoded amino acidthat comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.

As another example, a subject mitochondrial fission inhibitor constructor peptide linked to a water-soluble polymer can be made by reacting asubject mitochondrial fission inhibitor construct or peptide thatcomprises an alkyne-containing amino acid with a water-soluble polymer(e.g., PEG) that comprises an azide moiety; in some embodiments, theazide or alkyne group is linked to the PEG molecule through an amidelinkage. A “non-naturally encoded amino acid” refers to an amino acidthat is not one of the 20 common amino acids or pyrolysine orselenocysteine. Other terms that may be used synonymously with the term“non-naturally encoded amino acid” are “non-natural amino acid,”“unnatural amino acid,” “non-naturally-occurring amino acid,” andvariously hyphenated and non-hyphenated versions thereof. The term“non-naturally encoded amino acid” also includes, but is not limited to,amino acids that occur by modification (e.g. post-translationalmodifications) of a naturally encoded amino acid (including but notlimited to, the 20 common amino acids or pyrolysine and selenocysteine)but are not themselves naturally incorporated into a growing polypeptidechain by the translation complex. Examples of suchnon-naturally-occurring amino acids include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine.

In some embodiments, a subject mitochondrial fission inhibitor constructor peptide is linked (e.g., covalently linked) to a polymer (e.g., apolymer other than a polypeptide). Suitable polymers include, e.g.,biocompatible polymers, and water-soluble biocompatible polymers.Suitable polymers include synthetic polymers and naturally-occurringpolymers. Suitable polymers include, e.g., substituted or unsubstitutedstraight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymers or branched or unbranched polysaccharides, e.g.a homo- or hetero-polysaccharide. Suitable polymers include, e.g.,ethylene vinyl alcohol copolymer (commonly known by the generic nameEVOH or by the trade name EVAL); polybutylmethacrylate;poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone;poly(lactide-co-glycolide); poly(hydroxybutyrate);poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester;polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolicacid-co-trimethylene carbonate); polyphosphoester; polyphosphoesterurethane; poly(amino acids); cyanoacrylates; poly(trimethylenecarbonate); poly(iminocarbonate); copoly(ether-esters) (e.g.,poly(ethylene oxide)-poly(lactic acid) (PEO/PLA) co-polymers);polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin,fibrinogen, cellulose, starch, collagen and hyaluronic acid;polyurethanes; silicones; polyesters; polyolefins; polyisobutylene andethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinylhalide polymers and copolymers, such as polyvinyl chloride; polyvinylethers, such as polyvinyl methyl ether; polyvinylidene halides, such aspolyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile;polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinylesters, such as polyvinyl acetate; copolymers of vinyl monomers witheach other and olefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers. ABS resins, and ethylene-vinyl acetatecopolymers; polyamides, such as Nylon 66 and polycaprolactam; alkydresins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxyresins; polyurethanes; rayon; rayon-triacetate; cellulose; celluloseacetate; cellulose butyrate; cellulose acetate butyrate; cellophane;cellulose nitrate; cellulose propionate; cellulose ethers; amorphousTeflon; poly(ethylene glycol); and carboxymethyl cellulose.

Suitable synthetic polymers include unsubstituted and substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol), and derivatives thereof, e.g., substitutedpoly(ethyleneglycol) such as methoxypoly(ethyleneglycol), andderivatives thereof. Suitable naturally-occurring polymers include,e.g., albumin, amylose, dextran, glycogen, and derivatives thereof.

Suitable polymers can have an average molecular weight in a range offrom 500 Da to 50,000 Da, e.g., from 5000 Da to 40,000 Da, or from25,000 to 40,000 Da. For example, in some embodiments, where a subjectmitochondrial fission inhibitor construct or peptide comprises apoly(ethylene glycol) (PEG) or methoxypoly(ethyleneglycol) polymer, thePEG or methoxypoly(ethyleneglycol) polymer can have a molecular weightin a range of from about 0.5 kiloDaltons (kDa) to 1 kDa, from about 1kDa to 5 kDa, from 5 kDa to 10 kDa, from 10 kDa to 25 kDa, from 25 kDato 40 kDa, or from 40 kDa to 60 kDa.

Compositions as Modulators of Mitochondrial Fission and AssociatedActivities and Effects on Cells

Mitochondrial fission inhibitor peptides identified by the rationaldesign approach described above, are characterized and validated throughvarious functional assays described in more detail below. Thesefunctional assays are useful both in identifying inhibitors andactivators of mitochondrial fission and of abnormal cell activities, aswell as in characterizing the effects of the mitochondrial fissioninhibitor constructs and compositions on mitochondrial activities and onthe cells housing the mitochondria.

A mitochondrial fission peptide, or construct comprising the peptide,will have one or more of the following activities: 1) inhibition of Drp1GTPase activity; 2) inhibition of binding of Drp1 to Fis1; 3) reductionof mitochondrial damage in a cell under pathological conditions or otherconditions of stress; 4) reduction of cell death in a cell underpathological conditions or other conditions of stress; 5) reduction oftranslocation of Drp1 from the cytosol to a mitochondrion; 6) andinhibition of mitochondrial fragmentation in a cell under pathologicalconditions. Other effects include, but are not limited to, reducedmitochondrial fragmentation in neuronal cells exposed to severalmitochondrial toxins; reduced mitochondrial ROS(O₂—) production andsubsequently improved mitochondrial membrane potential and mitochondrialintegrity; increased cell viability through reduction in apoptosis andautophagic cell death; and reduced loss of neurites in primarydopaminergic neurons in a Parkinsonism cell culture model throughreduction in mitochondrial fragmentation and mitochondrial ROSproduction. In a preferred embodiment, treatment with or exposure to amitochondrial fission inhibitor construct or peptide will have minimaleffects on mitochondrial fission and cell viability of cells which arein non-stressed conditions or in a non-disease state.

In some embodiments, the inhibitor activity is selective, with respectto effects of the peptide or construct on a particular protein. In otherwords, a peptide or construct having selective inhibitory activity willinhibit the GTPase activity of Drp1 but inhibit the GTPase activity ofother proteins such as, but not limited to, MFN1 or OPA1. In otherembodiments, the inhibitor activity is selective in reducingmitochondrial damage, reducing cell death, reducing translocation ofDrp1 from the cytosol to a mitochondrion, or inhibiting mitochondrialfragmentation when used to treat a diseased or stressed cell as comparedto when the same inhibitor peptide or construct is used to treat ahealthy or non-stressed cell. For the purposes of the presentdisclosure, a diseased cell includes a healthy cell which has beentreated or genetically engineered to model a diseased cell.

To measure an increase or decrease of an activity or function upontreatment by a composition described herein, it is understood by theperson having ordinary skill in the art that the function or activitycan be measured, for example, in the presence and in the absence of thecomposition (e.g., mitochondrial fission inhibitor protein orconstruct), and a comparison is made between the levels of theactivities in the presence and absence of the composition.Alternatively, the function or activity can be measured, for example, inthe presence of two separate compositions, and the levels of theactivity or function in the presence of each composition are compared.An inhibition of an activity can be a reduction of about 5% to 10%, 5%to 20%, 2% to 20%, 10% to 20%, 5% to 25%, 20% to 50%, 40% to 60%, 50% to75%, 60% to 80%, 75% to 95%, 80% to 100%, 50% to 100%, 90% to 100%, or85% to 95% when comparing the two conditions. Similarly, activation ofan activity can be a increase of about 5% to 10%, 5% to 20%, 2% to 20%,10% to 20%, 5% to 25%, 20% to 50%, 40% to 60%, 50% to 75%, 60% to 80%,75% to 95%, 80% to 100%, 50% to 100%, 90% to 100%, 85% to 95%, or morethan 100% but less than 500%, when comparing the two conditions.

Methods of Making a Inhibitor Construct or Peptide

A mitochondrial fission inhibitor peptide or construct can be isolatedand purified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using high performance liquid chromatography (HPLC),exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. For the most part, the compositionswhich are used will comprise at least 80% by weight of the desiredproduct, at least about 85% by weight, at least about 95% by weight, orat least about 99.5% by weight, in relation to contaminants related tothe method of preparation of the product and its purification. Thepercentages can be based upon total protein.

A mitochondrial fission inhibitor peptide or construct may be preparedby in vitro (e.g., cell-free) synthesis, using conventional methods asknown in the art. Various commercial synthetic apparatuses areavailable, for example, automated synthesizers by Applied Biosystems,Inc., Foster City, Calif., Beckman, etc. By using synthesizers,naturally occurring amino acids may be substituted with unnatural aminoacids. The particular sequence and the manner of preparation will bedetermined by convenience, economics, purity required, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface, or provide some other desired property suchas increased solubility, increased resistance to proteolysis, increasedin vivo half-life, and the like. One or more cysteines can be used tomake thioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

A mitochondrial fission inhibitor peptide or construct as describedherein may be in the form of a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include acid addition salts, such ashydrochloride, hydrobromide, sulfurate, nitrate, phosphorate, acetate,propionate, glycolate, pyruvate, oxalate, malate, malonate, succinate,maleate, fumarate, tartarate, citrate, benzoate, cinnamate, mandelate,methanesulfonate, ethanesulfonate, p-toluene-sulfonate, salicylate andthe like, and base addition salts, such as sodium, potassium, calcium,magnesium, lithium, aluminum, zinc, ammonium, ethylenediamine, arginine,piperazine and the like.

Compositions

The present disclosure provides compositions comprising a mitochondrialfission inhibitor peptide or construct. The composition can comprise, inaddition to a mitochondrial fission inhibitor peptide or construct, oneor more of: a salt, e.g., NaCl, MgCl, KCl, MgSO₄, etc.; a bufferingagent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

Compositions comprising a mitochondrial fission inhibitor construct orpeptide may include a buffer, which is selected according to the desireduse of the peptide, and may also include other substances appropriate tothe intended use. Those skilled in the art can readily select anappropriate buffer, a wide variety of which are known in the art,suitable for an intended use.

In some cases, a mitochondrial fission inhibitor construct or peptidecomposition is a pharmaceutical composition. A subject pharmaceuticalcomposition can be administered to a subject in need thereof (e.g., asubject in need of inhibition of abnormal (e.g., pathological)mitochondrial fission). A subject pharmaceutical composition comprises:a) a mitochondrial fission inhibitor construct or peptide; and b) apharmaceutically acceptable excipient, a variety of which are known inthe art and need not be discussed in detail herein. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, “Remington: The Science andPractice of Pharmacy”, 19th Ed. (1995), or latest edition, MackPublishing Co; A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy”, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., edsed., Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.

Nucleic Acids Encoding the Constructs or Peptides

The present disclosure provides synthetic nucleic acids, where a subjectsynthetic nucleic acid comprises a nucleotide sequence encoding amitochondrial fission inhibitor peptide or construct. A nucleotidesequence encoding a mitochondrial fission inhibitor peptide or constructcan be operably linked to one or more regulatory elements, such as apromoter and enhancer, that allow expression of the nucleotide sequencein the intended target cells (e.g., a cell that is genetically modifiedto synthesize the encoded mitochondrial fission inhibitor construct orpeptide). In some embodiments, a subject nucleic acid is a recombinantexpression vector.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,cytomegalovirus immediate early promoter; herpes simplex virus thymidinekinase promoter; early and late SV40 promoters; promoters present inlong terminal repeats from a retrovirus; a metallothionein-1 promoter;and the like.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; promoters such as an ssaG promoter or a related promoter (see,e.g., U.S. Patent Publication No. 20040131637), a pagC promoter(Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the LacI repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.80:21-25).

A nucleotide sequence encoding a mitochondrial fission inhibitor peptideor construct can be present in an expression vector and/or a cloningvector. An expression vector can include a selectable marker, an originof replication, and other features that provide for replication and/ormaintenance of the vector.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant construct. Large numbers of suitable vectors andpromoters are known to those of skill in the art; many are commerciallyavailable for generating a subject recombinant constructs. The followingvectors are provided by way of example. Bacterial: pBs, phagescript,PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3,pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo,pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL(Pharmacia).

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a nucleic acidcomprising a nucleic acid sequence which encodes a mitochondrial fissioninhibitor peptide or construct. In some embodiments, a subject isolatedgenetically modified host cell can produce a mitochondrial fissioninhibitor construct or peptide.

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method.

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302.

Formulations, Dosages, and Routes of Administration

A mitochondrial fission inhibitor peptide or construct of the presentdisclosure (also referred to below as “active agent”) can beincorporated into a variety of formulations for therapeutic use (e.g.,for treating a subjection diagnosed with or suffering from a diseasewhich is associated with abnormal mitochondrial fission). Moreparticularly, a mitochondrial fission inhibitor peptide or construct canbe formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres,lotions, and aerosols. As such, administration of a mitochondrialfission inhibitor peptide or construct can be achieved in various ways,including oral, vaginal, buccal, rectal, parenteral, intraperitoneal,intravenous, intramuscular, intradermal, transdermal, intratracheal,etc., administration. A mitochondrial fission inhibitor peptide orconstruct can be systemic after administration or may be localized bythe use of an implant or other formulation that acts to retain theactive dose at the site of implantation.

A mitochondrial fission inhibitor peptide or construct can beadministered alone, in a combination of two or more mitochondrialfission inhibitor peptide or construct, or a mitochondrial fissioninhibitor peptide or construct can be used in combination with knowncompounds (e.g., therapeutic agents suitable for treating a diseaseassociated with abnormal mitochondrial fission, etc.) In pharmaceuticaldosage forms, a mitochondrial fission inhibitor peptide or construct maybe administered in the form of its pharmaceutically acceptable salt. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, a mitochondrial fission inhibitor peptide orconstruct can be used alone or in combination with appropriate additivesto make tablets, powders, granules or capsules, for example, withconventional additives, such as lactose, mannitol, corn starch or potatostarch; with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch or gelatins; with disintegrators, suchas corn starch, potato starch or sodium carboxymethylcellulose; withlubricants, such as talc or magnesium stearate; and if desired, withdiluents, buffering agents, moistening agents, preservatives andflavoring agents.

A mitochondrial fission inhibitor peptide or construct of the presentdisclosure can be formulated into preparations for injections bydissolving, suspending or emulsifying the peptide in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

A mitochondrial fission inhibitor peptide or construct of the presentdisclosure can be utilized in aerosol formulation to be administered viainhalation. A mitochondrial fission inhibitor construct or peptide ofthe present disclosure can be formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike.

A mitochondrial fission inhibitor peptide or construct of the presentdisclosure can be used in topical formulations, by formulation withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

Furthermore, a mitochondrial fission inhibitor peptide or construct ofthe present disclosure can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Amitochondrial fission inhibitor construct or peptide of the presentdisclosure can be administered rectally via a suppository. Thesuppository can include vehicles such as cocoa butter, carbowaxes andpolyethylene glycols, which melt at body temperature, yet are solidifiedat room temperature.

Unit dosage forms for oral, vaginal or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of a mitochondrial fission inhibitorpeptide or construct of the present disclosure. Similarly, unit dosageforms for injection or intravenous administration may comprise amitochondrial fission inhibitor peptide or construct in a composition asa solution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

Implants for sustained release formulations are well known in the art.Implants can be formulated as microspheres, slabs, etc. withbiodegradable or non-biodegradable polymers. For example, polymers oflactic acid and/or glycolic acid form an erodible polymer that iswell-tolerated by the host. An implant containing a mitochondrialfission inhibitor peptide or construct can be used, so that the localconcentration of active agent (mitochondrial fission inhibitor peptideor construct) is increased relative to the rest of the body.

Liposomes can be used as a delivery vehicle. The lipids can be anysuitable combination of known liposome forming lipids, includingcationic or zwitterionic lipids, such as phosphatidylcholine. Theremaining lipid can include neutral or acidic lipids, such ascholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Dosages

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and non-humananimal subjects, each unit containing a predetermined quantity of amitochondrial fission inhibitor peptide or construct calculated in anamount sufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the unit dosage forms depend on the particularpeptide or construct employed and the effect to be achieved, and thepharmacodynamics associated with the peptide or construct in the host.

Exemplary dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. An exemplarydosage may be one tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient. The time-releaseeffect may be obtained by capsule materials that dissolve at differentpH values, by capsules that release slowly by osmotic pressure, or byany other known means of controlled release.

Depending on the subject and condition being treated and on theadministration route, an active agent (e.g., a mitochondrial fissioninhibitor peptide or construct) may be administered in dosages of, forexample, 0.1 μg to 500 mg/kg body weight per day, e.g., from about 0.1μg/kg body weight per day to about 1 μg/kg body weight per day, fromabout 1 μg/kg body weight per day to about 25 μg/kg body weight per day,from about 25 μg/kg body weight per day to about 50 μg/kg body weightper day, from about 50 μg/kg body weight per day to about 100 μg/kg bodyweight per day, from about 100 μg/kg body weight per day to about 500μg/kg body weight per day, from about 500 μg/kg body weight per day toabout 1 mg/kg body weight per day, from about 1 mg/kg body weight perday to about 25 mg/kg body weight per day, from about 25 mg/kg bodyweight per day to about 50 mg/kg body weight per day, from about 50mg/kg body weight per day to about 100 mg/kg body weight per day, fromabout 100 mg/kg body weight per day to about 250 mg/kg body weight perday, or from about 250 mg/kg body weight per day to about 500 mg/kg bodyweight per day. The range is broad, since in general the efficacy of atherapeutic effect for different mammals varies widely with dosesgenerally being 20, 30 or even 40 times smaller (per unit body weight)in man than in the rat. Similarly the mode of administration can have aneffect on dosage. Thus, for example, oral dosages may be about ten timesthe injection dose. Higher doses may be used for localized routes ofdelivery.

A specific mitochondrial fission inhibitor peptide or construct can beadministered in an amount of from about 1 mg to about 1000 mg per dose,e.g., from about 1 mg to about 5 mg, from about 5 mg to about 10 mg,from about 10 mg to about 20 mg, from about 20 mg to about 25 mg, fromabout 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mgto about 200 mg, from about 200 mg to about 225 mg, from about 225 mg toabout 250 mg, from about 250 mg to about 300 mg, from about 300 mg toabout 350 mg, from about 350 mg to about 400 mg, from about 400 mg toabout 450 mg, from about 450 mg to about 500 mg, from about 500 mg toabout 750 mg, or from about 750 mg to about 1000 mg per dose.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific mitochondrial fission inhibitor peptide orconstruct, the severity of the symptoms and the susceptibility of thesubject to side effects. Some of the specific peptides may be morepotent than others. Preferred dosages for a given peptide are readilydeterminable by those of skill in the art by a variety of means. Onemeans is to measure the physiological potency of a given peptide.

Routes of Administration

An active agent (e.g., a mitochondrial fission inhibitor peptide orconstruct) is administered to an individual using any available methodand route suitable for drug delivery, including in vivo and ex vivomethods, as well as systemic and localized routes of administration.Administration can be acute (e.g., of short duration, e.g., a singleadministration, administration for one day to one week), or chronic(e.g., of long duration, e.g., administration for longer than one week,e.g., administration over a period of time of from about 2 weeks toabout one month, from about one month to about 3 months, from about 3months to about 6 months, or more).

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, transdermal, sublingual, topical application, intravenous,ocular (e.g., topically to the eye, intravitreal, etc.), rectal, nasal,oral, and other enteral and parenteral routes of administration. Routesof administration may be combined, if desired, or adjusted dependingupon the agent and/or the desired effect. A mitochondrial fissioninhibitor peptide or construct can be administered in a single dose orin multiple doses.

An active agent (e.g., a mitochondrial fission inhibitor peptide orconstruct) can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, and inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, ocular, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery ofthe agent. Where systemic delivery is desired, administration caninvolve invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

A mitochondrial fission inhibitor peptide or construct can also bedelivered to the subject by enteral administration. Enteral routes ofadministration include, but are not necessarily limited to, oral andrectal (e.g., using a suppository) delivery.

Methods of administration of a mitochondrial fission inhibitor peptideor construct through the skin or mucosa include, but are not necessarilylimited to, topical application of a suitable pharmaceuticalpreparation, transdermal transmission, injection and epidermaladministration. For transdermal transmission, absorption promoters oriontophoresis are suitable methods. Iontophoretic transmission may beaccomplished using commercially available “patches” which deliver theirproduct continuously via electric pulses through unbroken skin forperiods of several days or more.

Treatment Methods

The present disclosure provides methods for treating a subject sufferingfrom a disease or disorder characterized by, resulting from, orassociated with, abnormal mitochondrial fission, is provided, wherein amitochondrial fission inhibitor peptide or construct as described aboveis administered to the subject. Subjects amenable to treatment includeindividuals at risk of contracting the disease or disorder but notshowing symptoms, as well as subjects presently showing symptoms. Suchdiseases or disorders include neurodegenerative diseases, cardiacdiseases, mitochondriopathies, cancers, and the like. In someembodiments, the subject is suffering from Parkinson's disease,Huntington's disease, Alzheimer's disease, hypertension, encephalopathy,amyotrophic lateral sclerosis, cardiovascular disease, diabetes-inducedneuropathy, cardiopathy, ischemia, reperfusion injury, heart failure,peripheral artery disease, and cancer.

A therapeutically effective amount of a mitochondrial fission inhibitorpeptide or construct can be based on an amount of the peptide orconstruct which has been shown to be effective in reducing mitochondrialfission in a cell, for example, in vitro or in an animal model, comparedto the degree of mitochondrial fission observed in the same system inthe absence of treatment with the construct or peptide. The effectiveamount can be based on an amount which has been shown to be sufficientto reduce cell death (including, e.g., neuronal cell death) caused byabnormal mitochondrial fission at least about 10%, at least about 20%,at least about 30%, at least about 40%, at least about 50%, or more than50%, compared to the degree of cell death in the absence of treatmentwith the construct or peptide.

A method of the present disclosure for treating disorders and diseasescharacterized by, resulting from, or associated with, abnormalmitochondrial fission generally involves administering to a subject inneed thereof an effective amount of a mitochondrial fission inhibitorpeptide or construct. A therapeutically effective amount of amitochondrial fission inhibitor peptide or construct can be an amountsufficient to reduce an adverse symptom of the disease or disordercharacterized by, resulting from, or associated with, abnormalmitochondrial fission by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, or more than50%, compared to the degree of the adverse symptom in the absence oftreatment with the construct or peptide.

An effective amount of a mitochondrial fission inhibitor peptide orconstruct can be an amount sufficient to improve one or more functionsin an individual having a disease or disorder characterized by,resulting from, or associated with, abnormal mitochondrial fission,compared to the level of the function in the absence of treatment withthe peptide.

In some embodiments, multiple doses of an active agent are administered.The frequency of administration of a mitochondrial fission inhibitorpeptide or construct (“active agent”) can vary depending on any of avariety of factors, e.g., severity of the symptoms, etc. For example, insome embodiments, a mitochondrial fission inhibitor peptide or constructis administered once per month, twice per month, three times per month,every other week (qow), once per week (qw), twice per week (biw), threetimes per week (tiw), four times per week, five times per week, sixtimes per week, every other day (qod), daily (qd), twice a day (bid), orthree times a day (tid). As discussed above, in some embodiments, amitochondrial fission inhibitor peptide or construct is administeredcontinuously.

The duration of administration of a mitochondrial fission inhibitorpeptide or construct, e.g., the period of time over which amitochondrial fission inhibitor peptide or construct is administered,can vary, depending on any of a variety of factors, e.g., patientresponse, etc. For example, a mitochondrial fission inhibitor constructor peptide can be administered over a period of time ranging from aboutone day to about one week, from about two weeks to about four weeks,from about one month to about two months, or from about two months toabout four months, or more.

The activity of a mitochondrial fission inhibitor peptide or constructin reducing abnormal mitochondrial fission in a cell, and treating anindividual having a disease or disorder characterized by, resultingfrom, or associated with, abnormal mitochondrial fission, can be testedin a non-human animal model of such a disease or disorder.

In some aspects, a method for measuring the effects of a mitochondrialfission inhibitor peptide or construct when administered to an animalmodel is provided.

Suitable non-human animal models of Parkinson's disease (PD) include,e.g., the α-synuclein transgenic mouse model; and the1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) mouse model ofParkinson's disease. See, e.g., Betarbet et al. (2002) Bioessays 24:308;Orth and Tabrizi (2003) Mov. Disord. 18:729; Beal (2001) Nat. Rev.Neurosci. 2:325.

Suitable non-human animal models of Huntington's disease include, e.g.,a transgenic mouse comprising a human huntingtin transgene (e.g., the R6line, the YAC line), where the human huntingtin transgene comprises30-150 CAG repeats (encoding a polyglutamine expansion); a knock-inmouse model, comprising a homozygous or heterozygous replacement ofendogenous mouse huntingtin gene with a human huntingtin gene comprising30-150 CAG repeats. See, e.g., Mangiarini et al. (1996) Cell 87:493;Menalled (2005) NeuroRx 2:465; and Menalled and Chesselet (2002) TrendsPharmacol. Sci. 23:32; and Hodgson et al. (1999) Neuron 23:181.

For example, the effect of a mitochondrial fission inhibitor peptide oncognitive function, muscle function, motor function, brain function,behavior, and the like, is assessed. Electrophysiological tests can beused to assess brain function. Muscle function can be assessed using,e.g., a grip strength test. Motor function can be tested using, e.g., arotarod test. Cognitive functions can be tested using, e.g., the openfield test, the elevated plus maze, the Morris water maze, the zero mazetest, the novel objection recognition test, and the like. Tests forneurological functioning and behavior that include sensory and motorfunction, autonomic reflexes, emotional responses, and rudimentarycognition, can be carried out. Such tests are well known in the art;see, e.g., Chapter 12 “Assessments of Cognitive Deficits in Mutant Mice”by Rodriguiz and Wetsel, in “Animal Models of Cognitive Impairment”(2006) E. D. Levin and J. J. Buccafusco, eds. CRC Press, Boca Raton,Fla.

The effect of a mitochondrial fission inhibitor construct or peptide ondiabetes-induced neuropathy can be tested on a non-human animal model ofdiabetes (type 1 or type 2). See, e.g., Rees and Alcolado (2005) Diabet.Med. 22:359. Examples of suitable models include, e.g., the non-obesediabetic (NOD) mouse model. See, e.g., Kitukani and Makino (1992) Adv.Immunol. 51:285.

Subjects

Subjects suitable for treatment with a mitochondrial fission inhibitorpeptide or construct include, e.g., an individual who has been diagnosedas having a disorders and diseases characterized by, resulting from, orassociated with, abnormal mitochondrial fission, where such diseasesinclude, e.g., neurodegenerative diseases, cardiac diseases,mitochondriopathies, cancers, and the like. Such diseases include, butare not limited to, Parkinson's disease, Huntington's disease,Alzheimer's disease, hypertension, encephalopathy, amyotrophic lateralsclerosis, cardiovascular disease, diabetes-induced neuropathy,cardiopathy, ischemia, reperfusion injury, heart failure, peripheralartery disease, and cancer.

Inhibition of Drp1 GTPase Activity

In one embodiment, the fission inhibitor proteins and constructs asdescribed herein are identified by their ability to inhibit Drp1 GTPaseactivity. For example, the present disclosure describes how inhibitorproteins identified using the rational design approach described aboveare shown to be inhibitors of Drp1 GTPase activity. In anotherembodiment, the protein and construct is a selective inhibitor of theGTPase activity.

Drp1 is a large GTPase and its mitochondrial fission activity isdependent on its GTP hydrolysis (9). As described in Example 2, twofission constructs, P109 and P110, comprising amino acid sequencesderived from the Drp1 GTP exchange domain (GED) and GTPase domain,respectively (FIG. 1A), were tested for their ability to affect theenzymatic activity of Drp1. P109 and P110 inhibited 40% and 50% of theGTPase activity of recombinant Drp1, respectively (FIG. 2 a). Additionalexperiments showed, however, that P110 had no effect on the GTPaseactivity of other mitochondrial dynamics-related proteins, includingMFN1, OPA1, or Dynamin 1 (FIG. 2B). These data show that P110 comprisesa mitochondrial fission inhibitor peptide which selectively inhibitsDrp1 GTPase activity (e.g., inhibits Drp1 GTPase activity underconditions wherein P110 does not inhibit GTPase activity of a non-Drp1protein such as MFN1, OPA1 or Dynamin 1). In some embodiments, amitochondrial fission inhibitor construct comprises a mitochondrialfission inhibitor peptide which has a sequence which is about 42%, 57%,71%, or 86% identical to SEQ ID NO:12 (e.g., contains 1, 2, 3, or 4conservative amino acid substitutions such as Ser to Thr; e.g., SEQ IDNO:13), and in which the inhibitor construct or peptide selectivelyinhibits Drp1 GTPase activity. In other embodiments, the inhibitorconstruct comprises a sequence which is about 56%, 67%, 78%, or 89%identical to SEQ ID NO:13 or SEQ ID NO:16 (contains 1, 2, 3, or 4conservative amino acid substitutions), and in which the inhibitorconstruct or peptide inhibits Drp1 GTPase activity.

In some embodiments, a mitochondrial fission inhibitor construct orpeptide can inhibit GTPase activity of a Drp1 polypeptide by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or more than 80%, compared to the level of GTPase activity of the Drp1polypeptide in the absence of the mitochondrial fission inhibitorconstruct or peptide.

A mitochondrial fission inhibitor construct or peptide may have nosubstantial effect on GTPase activity of a polypeptide that has GTPaseactivity, other than a Drp1 polypeptide. Thus, a mitochondrial fissioninhibitor construct or peptide can in some cases selectively inhibitGTPase activity of a Drp1 polypeptide. For example, a mitochondrialfission inhibitor construct or peptide has no substantial effect onGTPase activity of mitofusin-1 or OPA1. Mitofusin-1 polypeptides areknown in the art. See, e.g., Santel et al. (2003) J. Cell Sci. 116:2763;Santel and Fuller (2001) J. Cell Sci. 114:867; Hales and Fuller (1997)Cell 90:121; and GenBank Accession No. NP_(—)284941. OPA1 polypeptidesare known in the art. See, e.g., Alexander et al. (2000) Nat. Genet,26:211; Dadgar et al. (2006) Exp. Eye Res. 83:702; and GenBank AccessionNo. NP_(—)056375.

In one embodiment, a method for administering a mitochondrial inhibitorconstruct or peptide to a cell in vitro or to an animal is provided,wherein the effects of the construct or peptide on the cell are measuredby measuring GTPase activity in the presence and in the absence of theinhibitor construct or peptide.

Inhibition of Drp1-Fis1 Interaction GTPase Activity

In one embodiment, the fission inhibitor proteins and constructs asdescribed herein are identified by their ability to inhibit interactionbetween Drp1 and Fis1. Activation of mitochondrial fission by Drp1involves the interaction of Drp1 with Fis1 which located in the outermembrane of the mitochondria. As shown in Example 2, the P110 inhibitorconstruct (SEQ ID NO:19) inhibited interaction of Drp1 and Fis1 onisolated mitochondria and in cultured neurons.

In some cases, a mitochondrial fission inhibitor construct or peptidecan inhibit binding of a Drp1 polypeptide to an Fis1 polypeptide by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or more than 80%, compared to the degree of binding of the Drp1polypeptide to the Fis1 polypeptide in the absence of the mitochondrialfission inhibitor construct or peptide.

In one embodiment, a method for administering a mitochondrial inhibitorconstruct or peptide to a cell in vitro or to an animal is provided,wherein the effects of the construct or peptide on the cell are measuredby measuring interaction of Drp1 and Fis1 on isolated mitochondria orcultured neurons.

Inhibition of Mitochondrial Fission

A mitochondrial fission inhibitor peptide or construct can reducemitochondrial fragmentation in a cell under pathological conditions(e.g., where mitochondria in the cell are undergoing pathologicalmitochondrial fission). For example, a mitochondrial fission inhibitorpeptide or construct can reduce mitochondrial fragmentation in a cell byat least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or more than 80%, compared to the degree of mitochondrialfragmentation in the absence of the mitochondrial fission inhibitorconstruct or peptide.

In one embodiment, a method for administering a mitochondrial inhibitorconstruct or peptide to a cell in vitro or to an animal is provided,wherein the effects of the construct or peptide on the cell are measuredby measuring the level of mitochondrial fragmentation in a cell (e.g.,cultured neurons) which was treated in vitro or which was isolated froman animal which had been administered the construct or peptide.

Inhibition of Drp1 Translocation to the Mitochondria

A mitochondrial fission inhibitor peptide or construct can in some casesinhibit translocation of a Drp1 polypeptide from the cytosol tomitochondria in a cell. For example, a mitochondrial fission inhibitorpeptide or construct can inhibit translocation of a Drp1 polypeptidefrom the cytosol to mitochondria in a cell by at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or more than80%, compared to the degree of translocation of the Drp1 polypeptidefrom the cytosol to mitochondria in the absence of the mitochondrialfission inhibitor peptide or construct.

In one embodiment, a method for administering a mitochondrial inhibitorconstruct or peptide to a cell in vitro or to an animal is provided,wherein the effects of the construct or peptide on the cell are measuredby measuring the translocation of Drp1 to mitochondria in a cell (e.g.,cultured neurons) which was treated in vitro or which was isolated froman animal which had been administered the construct or peptide.

Effects on Mitochondrial Morphology

The mitochondrial fission inhibitor constructs or peptides as describedherein can affect mitochondrial morphology. More specifically, theconstructs or peptides may function to decrease the extent ofmitochondrial fragmentation as viewed using confocal microscopy with theappropriate fluorescent-labeled markers as described in the Examplesbelow. In preferred embodiments, the inhibitor constructs or peptidesdescribed herein decrease the extent of mitochondrial fragmentation in acell which is under a stressed condition as compared to effects of theconstruct or peptide in a cell which is not under a stressed condition.In some embodiments, the cell is a neuronal cell.

Effects on Production of Reactive Oxygen Species (ROS) and MembranePotential

The fission inhibitor constructs or peptides as described herein canaffect the production of reactive oxygen species (ROS) by a cell.Reactive oxygen species include, but are not limited to, hydrogenperoxide. The constructs or peptides can reduce production ofmitochondrial superoxide or can reduce or inhibit cytochrome c releaseby the mitochondria. As noted above, such effects of the peptide orconstructs are preferably observed in cells which are stressed, but theeffects are not detected, or occur to a much lesser extent in cellswhich are not stressed. Similarly, the constructs or peptides describedherein can improve or increase mitochondrial membrane potential (MMP) instressed cells.

In one embodiment, a method for administering a mitochondrial inhibitorconstruct or peptide to a cell in vitro or to an animal is provided,wherein the effects of the construct or peptide on the cell ormitochondria obtained from the cell result in a decrease in theproduction of one or more reactive oxygen species, or result in anincrease in mitochondrial membrane potential. In another embodiment, theeffects are determined in stressed and/or diseased cells and thedecrease in ROS or increase in MMP is relative to ROS production or MMP,respectively, in non-stressed and/or healthy cells.

Effects on Programmed Cell Death and Cell Survival

The fission inhibitor constructs or peptides as described herein canreduce the onset of programmed cell death and increase cell survival. Inone embodiment, a method for administering a mitochondrial inhibitorconstruct or peptide to a cell in vitro or to an animal is provided,wherein the effects of the construct or peptide on the cell ormitochondria obtained from the cell result in a decrease in the onset ofprogrammed cell death as measured by reduced accumulation of active Baxon the mitochondria, reduced or blocked release of cytochrome c from themitochondria and increased Bcl-2 levels on the mitochondria, as comparedto cells which have not been treated with or exposed to the inhibitorconstruct or peptide. In another embodiment, cells or animals which areexposed to or treated with the inhibitor construct or peptide exhibit areduction in the number of cells which are Annexin V-positive ascompared to cells which were not exposed to or treated with theinhibitor construct or peptide.

Effects on Neurite Degeneration in a Parkinsonism Model

The fission inhibitor constructs or peptides as described herein canreduce the neuronal degeneration in a cell culture model ofParkinsonism. In one embodiment, a method for administering amitochondrial inhibitor construct or peptide to a cell in vitro or to ananimal is provided, wherein the effects of the construct or peptide onthe cell or mitochondria obtained from the cell result in reducedneurite loss of dopaminergic neurons as measured by levels of tyrosinehydroxylase (TH), as compared to cells which have not been treated withor exposed to the inhibitor construct or peptide.

Although the mitochondrial fission inhibitor constructs described hereinmay be peptides, such peptides may be bioavailable. For example, many ofthe constructs described herein are linked to a cell-permeable peptidecarrier (e.g., TAT₄₇₋₅₇). Linking a biologically active peptide to sucha carrier peptide or other transduction moiety as described herein hasbeen widely and successfully used for cargo delivery in cultures (invitro) and in vivo. Previous studies showed that TAT-conjugated peptidescan quickly enter cells and pass through the blood-brain-barrier (43,46)and have extensive bio-distribution within minutes after single dosetreatment in vivo and in cultures (47,48). Thus, P110 may be useful intreatment of diseases associated with excessive mitochondrial fission,such as Parkinsonism.

Developing strategies to limit mitochondrial damage and to ensurecellular integrity by inhibiting mitochondrial fission impairment canidentify important new therapeutics. The first inhibitor ofmitochondrial fission, Mdivi-1, was identified using a chemical screenin yeast cells harboring a mitochondrial fusion-defective mutant fzo1-1(human mitofusin 1) (49). Mdivi-1 increases growth rate in yeast andinhibits mitochondrial division in yeast and mammals by blocking Dnm1(yeast Drp1) polymerization. However, whether Mdivi-1 affects mammalianDrp1 in the same way as in yeast Dnm1 remains to be determined.

As described herein, Drp1-induced mitochondrial ROS disrupted themitochondrial membrane potential (MMP), an important step for theinduction of mitochondria-mediated cell death (Twig et al., 2008, EMBOJ. 27:433-446). These data are in agreement with the report thatmitochondrial ROS is an upstream of MMP reduction, which is essentialfor initiation of mitochondrial fission. Indeed, it was discovered thata mitochondrial fission inhibitor construct, e.g., P110, can inhibitexcessive mitochondrial fission in the presence of stressors, such asMPP+ and COOP, resulting in decreased ROS production, preservation ofmitochondrial membrane potential and inhibition of cell death. Excessivemitochondrial fission and fragmentation leads to apoptosis and autophagyin a number of human cell lines, such as human SH-SY5Y neuronal cells(Wang et al., 2011, Aging Cell 10:807-823). Drp1 function is requiredfor apoptotic mitochondrial fission, as either expression of adominant-negative mutant (Drp1K38A) or downregulation of Drp1 by RNAidelay mitochondrial fragmentation, cytochrome c release, caspaseactivation, and cell death (Cassidy-Stone et al., 2008, Dev Cell14:193-204; Cereghetti et al., 2010, Cell Death Differ 17:1785-1794).Consistent with previous studies, it is shown herein that inhibition ofDrp1 with a mitochondrial fission inhibitor construct, e.g., P110,prevents the accumulation of the apoptotic factor, Bax, on themitochondria and cytochrome c release, and can recover anti-apoptoticfactor Bcl-2 levels. The changes of these Bcl-2 family proteins andinhibition of the consequent cytochrome c release reflect a correctionof the compromised MMP, which causes apoptosis at a later stage.Further, fission can yield asymmetric daughter mitochondria that differin membrane potential (Rivolta et al., 2002, Brain Res Dev Brain Res133:49-56), which could be later targeted by the autophagy machinery(Twig et al., 2008, EMBO 27:433-446). Indeed, overexpression of Fis1reduces mitochondrial mass and triggers autophagy (Gomes et al., 2008,Biochim Biophys Acta 1777:860-866), whereas overexpression of a Drp1dominant negative mutant reduces autophagy (Barsoum et al., 2006, EMBOJ. 25:3900-3911). Because a Drp1 peptide inhibitor, e.g., P110, greatlyreduces MPP+-induced autophagy and apoptosis, it is likely thatexcessive activation of Drp1-mediated mitochondrial fission is involvedin executing multiple cell death pathways (Reddy et al., 2011, Brain ResRev 67:103-118). Importantly, inhibition of Drp1 by P110 could reducethese types of cell death at initial stage of mitochondrial damages,thus leading to neuronal cell protection.

A causal role of Drp1-dependent mitochondrial fission impairment in thepathogenesis of Parkinsonism has been recently reported (Bueler, 2009,218:235-246). The Parkinsonism-inducing neurotoxins, 6-hydroxy dopamine,rotenone, and MPP+, all trigger Drp1 translocation to the mitochondriaand mitochondrial fragmentation (fission), thus leading to dopaminergiccell death in neuronal cultures. Parkinsonism-related proteins PINK1,parkin, DJ-1 and alpha-synuclein appear to control mitochondrialfunction by associating with Drp1 and regulating mitochondrialfusion/fission events. It is clear that neurotoxins causing Parkinsonismand Parkinsonism-associated genes are related to mitochondrial dynamics.Thus, controlling Drp1-mediated mitochondrial fission impairment inParkinsonism may be of particular importance to inhibitneurodegeneration. Using cell culture models of Parkinsonism, it wasdetermined as described herein that in response to MPP+, a mitochondrialfission inhibitor construct or peptide, e.g, P110, reduces dopaminergicneuronal degeneration by inhibiting Drp1-mediated mitochondrialdysfunction. In addition, impaired mitochondrial dynamics and excessivemitochondrial fission have been connected to a number of neurologicaldisorders, including neurodegenerative diseases (Knott et al., 2008, NatRev Neurosci 9:505-518), hypertensive encephalopathy (Qi et al., 2008, JClin Invest 118:173-182), stroke (Liu et al., 2012, Brain Res1456:94-99) and neurologic pain (Ferrari et al., 2011, J Neurosci31:11404-11410). Therefore, an inhibitor of Drp1, such as P110, may be auseful treatment for the diseases in which impairment of mitochondrialdynamics occurs.

Further, using a model of Parkinson's disease (PD) in culture, it wasdemonstrated herein that a mitochondrial fission inhibitor construct orpeptide, e.g, P110, is neuroprotective by inhibiting mitochondrialfragmentation and ROS production and subsequently improvingmitochondrial membrane potential and mitochondrial integrity. Amitochondrial fission inhibitor construct or peptide, e.g, P110, canincrease neuronal cell viability by reducing apoptosis and autophagiccell death, and reducing neurites loss of primary dopaminergic neuronsin this PD cell culture model. Importantly, it was also discovered thattreatment with a mitochondrial fission inhibitor construct or peptide,e.g, P110, had minimal effects on mitochondrial fission and cellviability under normal conditions.

FURTHER EMBODIMENTS

Embodiment 1 is a mitochondrial fission inhibitor construct comprising amitochondrial fission inhibitor peptide comprising about 7 to 20 aminoacids, wherein the peptide comprises an amino acid sequence having atleast about 80% amino acid identity to a contiguous stretch of fromabout 7 to 20 amino acids of a Drp1 polypeptide (SEQ ID NO:1) or a Fis1polypeptide (SEQ ID NO:2).

Embodiment 2 is the inhibitor construct of embodiment 1, furthercomprising a protein transduction moiety.

Embodiment 3 is the inhibitor construct of embodiment 2, wherein theprotein transduction moiety is a carrier peptide

Embodiment 4 is the inhibitor construct of embodiment 2 or 3, whereinthe protein transduction moiety comprises a carrier peptide derived froma human immunodeficiency virus Tat polypeptide.

Embodiment 5 is the inhibitor construct of embodiment 2, 3 or 4, whereinthe protein transduction moiety comprises SEQ ID NO:32.

Embodiment 6 is the inhibitor construct of embodiment 1, wherein theconstruct is a linear peptide comprising:

-   -   a) the fission inhibitor peptide;    -   b) an optional linker; and    -   c) a carrier peptide.

Embodiment 7 is the inhibitor construct of embodiment 6, comprising alinker, wherein the linker is positioned between the fission inhibitorpeptide and the carrier peptide.

Embodiment 8 is the inhibitor construct of any one of embodiments 1-7,wherein the fission inhibitor peptide comprises SEQ ID NO:12.

Embodiment 9 is the inhibitor construct of embodiment 6 or 7, comprisingSEQ ID NO:19.

Embodiment 10 is the inhibitor construct of any one of embodiments 1-9,wherein the inhibitor construct selectively inhibits a GTPase activityof the Drp1 polypeptide.

Embodiment 11 is the inhibitor construct of any one of embodiments 1-9,wherein the inhibitor construct inhibits binding of the Drp1 polypeptideto the Fis1 polypeptide.

Embodiment 12 is the inhibitor construct of any one of embodiments 1-9,wherein the inhibitor construct reduces mitochondrial fragmentation in acell.

Embodiment 13 is the inhibitor construct of any one of embodiments 1-9,wherein the inhibitor construct inhibits translocation of the Drp1polypeptide from the cytosol to a mitochondrion in a cell.

Embodiment 14 is the use of the inhibitor construct of any one ofembodiments 1-13 for inhibiting or reducing abnormal mitochondrialfission, comprising contacting a cell with a composition comprising amitochondrial fission inhibitor construct, wherein the contactingreduces abnormal mitochondrial fission.

Embodiment 15 is the use according to embodiment 14, wherein thecontacting a cell comprises administering the composition to an animal.

Embodiment 16 is the use according to embodiment 14 or 15, whereinadministering the composition to the animal results in a decrease intremor, bradykinesia, rigidiy, and/or postural dysfunction.

Embodiment 17 is the use of the inhibitor construct of any one ofembodiments 1-13 for treating a subject suffering from, diagnosed withor predisposed to a disease or disorder associated with abnormalmitochondrial fission, comprising administering to a subject having ordiagnosed with the disease or disorder a therapeutically effectiveamount of a pharmaceutical composition comprising a mitochondrialfission inhibitor construct, wherein the administering is effective toreduce at least one adverse symptom of the disease.

Embodiment 18 is the use according to embodiment 17, wherein theadministering is by a route selected from oral, intravenous,intramuscular, and subcutaneous.

Embodiment 19 is the use according to embodiment 17 or 18, wherein thedisease or disorder is Parkinson's disease, Huntington's disease,Alzheimer's disease, ischemia, reperfusion injury, diabetes-inducedneuropathy, or heart disease.

Embodiment 20 is the use according to embodiment 17, 18 or 19, whereinadministering the composition to the animal results in a decrease intremor, bradykinesia, rigidly, and/or postural dysfunction.

Ranging from tool design, target validation and bio-efficacydemonstration, the findings from the studies described herein are noveland important for understanding the role of mitochondrial impairment inneurodegeneration as well as representing a general and easy approach toidentify protein-protein interaction inhibitors. Inhibitors, such aspeptide inhibitor P110, open up a therapeutic avenue for treatment ofneurodegenerative diseases, such as Parkinson's disease, which has noeffective treatment available.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Rational Design of Modulator Peptides

An L-ALIGN sequence alignment software 31 was used, resulting in theidentification of 3 different regions of similarity between Drp1 (humanDrp1, GenBank Acc. No. AAH24590) and F is 1 (human Fis1, GenBank Acc.No. NP_(—)057152). The regions of similarity are shown as a schematic inFIG. 1A, marked as regions 108-113. Crystal structures for theseproteins are known and show that these regions are present on thesurface of Drp1 and Fis1, thus likely accessible for protein-proteininteraction (PPI). Further, all these sequences are conserved in avariety of species (FIG. 10). However, only the sequence in region 110is conserved in mammals, fish, chicken and yeast, suggesting that thisregion is most likely critical for the function of Drp1. Furthersequence alignment analysis showed that there are 17 other proteins inthe human genome which contain a sequence that is at least 80% similarto the sequence in region 110. These proteins include TOM22, DYN1, DYN2,DYN3, MIA3, SCN5A, HIP1, PCDGK, BI2L2, ZSWIM5, ADAM 17, AP2B, ZSWM4,CYP2W1 and MSLN. However, Fis1 was the only protein in which thissequence was 100% identical in other mammals, further supporting theidea that 110 represents an important region within Drp1 for interactionwith Fis1.

Peptides corresponding to region 110, as well as to the five othersequence similarity regions which had been identified between Fis1 andDrp1 were synthesized using a standard phase peptide synthetic method.Peptides were synthesized using a microwave by Liberty Microwave PeptideSynthesizer (CEM Corporation, Matthews, N.C., USA). The C-terminus ofthe peptides was modified to C(O)—NH₂ using Rink Amide AM resin toincrease stability (Gomes and Scorrano, 2008, Biochim Biophys Acta1777:860-866). Peptides were analyzed by analytical reverse-phasehigh-pressure liquid chromatography (RP-HPLC) (Shimadzu, Md., USA) andmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MS) and purified by preparative RP-HPLC (Shimadzu, Md., USA).

Each peptide was linked via a Gly-Gly (GG) linker to the cell permeatingTAT protein-derived peptide, TAT₄₇₋₅₇, (Chen et al., 2001, Chem Biol8:1123-1129; Zhu et al., 2004, J Biol Chem 279:35967-35974) to produce asingle linear peptide. These mitochondrial fission inhibitor constructsare referred to herein as P110, P108, P109, P111, P112 and P113:

The constructs used are as follows:

(SEQ ID NO: 17) P108: RRRQRRKKRGYGGSTQELLRFPK; (SEQ ID NO: 18) P109:RRRQRRKKRGYGGKLSAREQRD; (SEQ ID NO: 19) P110: RRRQRRKKRGYGGDLLPRGS;(SEQ ID NO: 20) P110a: RRRQRRKKRGYGGDLLPRGT; (SEQ ID NO: 21) P111:RRRQRRKKRGYGGCSVEDLLKFEK; (SEQ ID NO: 22) P112: RRRQRRKKRGYGGKGSKEEQRD;and (SEQ ID NO: 23) P113: RRRQRRKKRGYGGELLPKGS.

Example 2 Effects on Drp1 GTPase Activity In Vitro

First, mitochondrial fission inhibitor constructs were tested todetermine what, if any, effects each peptide had on enzyme (GTPase)activity. Recombinant protein Drp1, Mfn1, OPA1 or dynamin-1 (25 ng) wasincubated with the indicated modulator peptides for 30 min. GTPaseactivity was assayed in vitro using a GTPase assay kit (InnovaBiosciences, Littleton, USA, or Novus Biologicals, Littleton, Colo.)according to manufacturer's instructions.

In an in vitro GTPase assay, two of the peptide inhibitor constructs(P110 and P112) specifically inhibited Drp1 GTPase activity by 30% and50% (n=3), respectively. These two inhibitor constructs had no effectson the GTPase activity of mitofusin1 and OPA1. In the presence of P109and P110, a 40% and 50% of inhibition in the GTPase activity,respectively, was observed (FIG. 2A). The other peptides, including theconstructs comprising the corresponding homologous peptides derived fromFis1, P112 and P113, respectively, exerted no significant effect. Thesedata are surprising, because they indicate that these two Drp1-derivedpeptides interact with Drp1, thus suggesting that the sequencecorresponding to P109 and P110 are also involved in intra-molecularinteractions or in inter-molecular interactions between oligomers ofDrp1 (Zhu et al., 2004, J Biol Chem 279:35967-35974). A dominantnegative mutant of Drp1, Drp1K38A, which inhibits Drp1 GTPase activityand fission activity (Frank et al., 2001, Dev Cell 1:515-525), was usedas a positive control and reduced Drp1 activity by ˜70% (FIG. 2A).Importantly, peptide P110 had no effects on the GTPase activities ofother mitochondrial dynamics-related proteins, such as Mfn1 and OPA1(FIG. 2B). P110 had also no effects on the GTPase activity of therelated protein, dynamin-1 (FIG. 2B), which belongs to DRP family andmediates endocytosis of the plasma membrane (McClure and Robinson, 1996,Mal Membr Biol 13:189-215). These data indicate that peptide P110 isselective for Drp1. In contrast, P109 increased the GTPase activity ofMfn1 and OPA1 in the same assay (MFN1, 229%±10; OPA1, 177%±13; p<0.05,n=3, respectively (FIG. 2F), indicating that only P110 is a selectivefor Drp1.

Example 3 Interaction Between Drp1 and Fis1

If the peptides designed do represent PPI surfaces between Drp1 andFis1, the peptides should inhibit the association of Drp1 with themitochondria. To obtain isolated mitochondria, SH-5YSH cells were washedwith cold phosphate-buffered saline (PBS) and incubated on ice in lysisbuffer (250 mM sucrose, 20 mmol/L HEPES-NaOH, pH 7.5, 10 mmol/L KCl, 1.5mmol/L MgCl₂, 1 mmol/L EDTA, protease inhibitor cocktail, phosphataseinhibitor cocktail) for 30 minutes. Cells were scraped and thendisrupted 10 times by repeated aspiration through a 25 gauge needle,followed by a 30 gauge needle. Mouse liver tissue was minced and groundby pestle in lysis buffer. The homogenates were spun at 800 g for 10 minat 4° C. and the resulting supernatants were spun at 10,000 g for 20minutes at 4° C. The pellets were washed with lysis buffer and spun at10,000 g again for 20 minutes at 4° C. The final pellets were suspendedin lysis buffer containing 1% Triton X-100 and were mitochondrial-richlysate fractions.

SH-SY5Y cells were treated with Drp1 peptides P110, P109, P112 orTAT₄₇₋₅₇ only (1 μM). Because the interaction between Drp1 and Fis1 istransient and dynamic, cells were treated with the crosslinker, DSP,(0.75 mM for 30 min), as described in a previous study (Yoon et al.,2003, Mol Cell Biol 23:5409-5420). The cells were lysed in total celllystes (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% Triton X-100,and protease inhibitor). Immunoprecipitates were analyzed by SDS-PAGEand immunoblotting with antibodies to Drp1, VDAC, and Fis1. VDAC wasused as a loading control

Mitochondria isolated from mouse liver were incubated with recombinantDrp1 (GST-Drp1). Drp1 bound to mitochondrial preparation and only P110(1 μM) reduced this association by >60% (p<0.01, FIG. 2C); peptidecarrier TAT₄₇₋₅₇ or the other Drp1-derived peptides had no effect. Themitochondrial membrane protein VDAC was used as marker and loadingcontrol.

In another set of experiments, Drp1 was incubated with Fis1 and thecomplex was immunoprecipitated using anti-Drp1 antibody followed byimmunoblotting with anti-Fis1 antibody. For direct binding between Drp1and Fis1, recombinant Drp1 (50 ng) and Fis1 (50 ng) were incubated inPBS containing 1 mM DSP for 30 min in the presence or absence ofDrp1/Fis1 peptides (1 μM each) or control peptide TAT. After terminationof the reaction by 50 mM Tris-HCl, pH 7.5, reaction mixtures weresubjected to immunoprecipitation with anti-Fis1 antibody in the presenceof 1% Triton X-100. Immunoprecipitates were washed with PBS containing1% Triton X-100 and analyzed by SDS-PAGE and subsequent immunoblottingwith antibodies to Fis1 and Drp1.

Drp1 bound Fis1 in this assay and this interaction was blocked by theaddition of P110 but not by other peptides (FIG. 2D). Inhibition of theinteraction between Drp1 and Fis1 by peptide P110 was also observed inthe cultured SH-SY5Y neuronal cells (FIG. 2E). Again, other peptideshave no effect on blocking the interaction.

P110 was further characterized to determine its effect on Drp1 activityand functions in cell culture.

Example 4 Effects of Mitochondrial Fission Inhibitor Construct on Drp1Translocation

Drp1 translocation from cytosol to the mitochondria is a hallmark ofmitochondrial fission. Experiments were done to determine if P110 caninhibit translocation of Drp1 to the mitochondria in cultured humanneuroblastoma, SH-SY5Y cells.

Cultured human SH-SY5Y neuronal cells were treated with peptide P110 (1μM) for 30 min prior to 1 hr incubation in the absence or presence ofMPP+ (1-methyl-4-phenylpyridinium, a specific mitochondrial complex Iinhibitor) (2 mM) or CCCP (carbonyl cyanide m-chloro phenyl hydrazone)(10 μM). Western blot analysis of mitochondrial fractions was determinedby the indicated antibodies. VDAC was used as a loading control.

SH-SY5Y cells were treated with MPP+, a neurotoxin causing PD, CCCP, amitochondrial uncoupler (FIG. 3A), H₂O₂ (hydrogen peroxide, oxidativestress, FIG. 3B) or rotenone (a mitochondrial complex I inhibitor and aneurotoxin causing PD, FIG. 3B). Treatment with P110 abolished theincreased levels of Drp1 associated with the mitochondrial fractionsunder all these stress conditions, suggesting that P110 blocked Drp1translocation to the mitochondria induced by a wide range of stimuli.

In another experiment, mitochondria were isolated from SH-SY5Y neuronalcells exposed to MPP+ in the absence or presence of P110. Mitochondrialfractions were subjected to Western blot analysis. The levels of Drp1,Fis1, Mff, Mfn1 (mitofisin1) and OPA1 in the mitochondrial fractionswere determined. Voltage-dependent ion channel (VDAC), a marker ofmitochondria, was used as internal loading controls. Quantitative datafrom three independent experiments are provided a the histogram, withdata shown as mean±S.E (FIG. 3D).

Treatment with P110 reduced MPP+-induced translocation of Drp1 fromcytosol to the mitochondria by more than 60% (p<0.01, n=3), but did notaffect the cellular level and distribution of other mitochondrialdynamics-related proteins, including mitofusin-1 and -2 (MFN-1/2), OPA1and Fis1 (FIGS. 3C and 3D).

The function of Drp1 is regulated at least in part by phosphorylationevents. Previous studies demonstrated that Drp1 phosphorylation atSer616 results in increased Drp1 fission activity and promotesmitochondrial fragmentation (Qi et al., 2011, Mol Biol Cell 22:256-265).As shown in FIG. 3F, P110 treatment abolished Ser616 phosphorylation incultured SH-SY5Y neuronal cells exposed to either MPP+ or CCCP. Notethat treatment with P110 did not affect Drp1 levels and phosphorylationunder normal conditions (FIGS. 3A and 3B) and that no changes in thetotal levels of Drp1 or other proteins associated with mitochondrialfusion and fission were observed in any of the treatment groups (FIG.3E). Together these data confirm selectivity of P110 for Drp1.

The effects of MPP+ or CCCP on translocation of Drp1 in the absence andpresence of P110 was measured using confocal microscopy. CulturedSH-SY5Y cells in black 96-well plate were treated with Drp1 peptideinhibitor P110 (1 μM) 30 min prior to treatment of MPP+ (2 mM) or CCCP(5 uM). After 2 his of treatment, cells were stained with MITOSOX™ (redmitochondrial superoxide indicator) (5 uM, for 10 min, mitochondrialROS) or TMRM (0.5 uM, for 20 min, MMP) at 37° C. For total ROSdetection, cells were treated with MPP+ or CCCP for 24 hrs. ROS levelwas measured by CM-H2DCFDA (1 μM for 30 min, Invitrogen). Cells werewashed with PBS for three times. The detection was performed in afluorescence microplate reader (Tecan, Infinite M1000) according tomanufacturer's instructions. Measurements were normalized to the cellnumber counted using Hoechst staining.

Confocal imaging analysis demonstrated that Drp1 is localized on themitochondria after SH-SY5Y neuronal cells were exposed to MPP+ or CCCP.By contrast, P110 treatment greatly reduced this co-localization. Thedata, quantitated by Image J software, is provided in a histogram FIG.3G. These results show that peptide P110 inhibited Drp1 association withthe mitochondria as well as the enzymatic activity of Drp1, and supportthe conclusion that peptide P110 is a specific inhibitor Drp1.

Example 5 Effects of Modulator Peptides on Mitochondrial Morphology

To determine effects of P110 on mitochondrial morphology in response tomitochondrial stresses, an immuno-staining approach was used. In oneexperiment, SH-SY5Y neuronal cells were treated with Drp1 peptides orDrp1 siRNA followed by treatment of MPP+. The cells were then stainedwith TOM20 and Hoechst stain. As presented in FIGS. 4A and 4B, (thepercentage of cells with fragmented mitochondria relative to the totalnumber of cells presented as the mean±S.E. of three independentexperiments. **, p<0.01; at least 200 cells per group were counted),treatment with P109 and P110 reduced mitochondrial fragmentation in thecultured cells exposed to MPP+ by 70%, (p<0.01, n=3), similar to thereduction achieved by Drp1 siRNA treatment under the same condition.Importantly, treatment with these novel peptide inhibitors had no effecton mitochondrial morphology in control cells.

In another experiment, cultured SH-SY5Y cells were treated with P110 (1μM) for 30 min followed by incubation with MPP+ (2 mM, 4 hrs) or CCCP (5uM, 30 min). The cells were then stained with anti-Tom 20 antibody(green) and Hoechst stain (scale bar 0.5 μm). Mitochondrial morphologywas analyzed using 63× oil lens. The data were analyzed and arepresented as a histogram in which the percentage of cells withfragmented mitochondria relative to the total number of cells ispresented as the mean±S.E. of 3 independent experiments. At least 200cells per group were counted.

As depicted in FIG. 5, extensive mitochondrial fragmentation wasobserved. The fragmentation visualized by confocal microscopy wasfeatured by small, round or dot-like staining pattern in the culturesexposed to MPP+ or COOP treatments, indicating that mitochondrialnetwork was disrupted and fragmented. By contrast, mitochondrialfragmentation was greatly reduced by the treatment with Drp1 peptideinhibitor P110 in cells exposed to MPP+ (50% to 14%, p<0.05) and CCCP(63% to 23%, p<0.05), respectively. The extent of inhibition ofmitochondrial fragmentation by P110 treatment was similar to that fromthe group treated with Drp1 siRNA under the same conditions.Interestingly, in the control cells incubated with P110, we did notobserve dramatic difference on mitochondrial network as compared withcontrol cells (FIG. 5). Given that P110 treatment did not affect Drp1under normal conditions, we proposed that the peptide inhibitor P110might have greater impact on Drp1 under pathological conditions in whichDrp1 is hyper-activated.

Example 6 Effects on Mitochondrial ROS Production

Drp1-dependent mitochondrial fission impairment has been shown to occurduring the early stage of mitochondrial dysfunction (Barsoum et al.,2006, EMBO J. 25:3900-3911; Yuan et al., 2007, Cell Death Differ14:462-471). Experiments were done to determine if P110 affectsmitochondrial dysfunction as measured by the production of reactiveoxygen species (ROS) under stress conditions and the effects on membranepotential.

SH-SY5Y cells were treated with P110 (1 μM) followed by incubation ofMPP+ (2 mM) for 2 hrs. Mitochondrial superoxide production wasdetermined using the mitochondrial superoxide indicator, MITOSOX™ red(red mitochondrial superoxide indicator). Nuclei were stained withHoechst (blue). This experiment included the analysis of P110alanine-scan analogs, further determine the contribution of each of theamino acids of the P110 fission inhibitor peptide sequence on thebioactivity of the peptide. The resultant sequences are shown here:

YGRKKRRQRRRGGaLLPRGS (SEQ ID NO: 24) YGRKKRRQRRRGGDaLPRGS(SEQ ID NO: 25) YGRKKRRQRRRGGDLaPRGS (SEQ ID NO: 26)YGRKKRRQRRRGGDLLaRGS (SEQ ID NO: 27) YGRKKRRQRRRGGDLLPaGS(SEQ ID NO: 28) YGRKKRRQRRRGGDLLPRaS (SEQ ID NO: 29)YGRKKRRQRRRGGDLLPRGa. (SEQ ID NO: 30)

The data are shown as a quantitative histogram of red fluorescence (FIG.6A). P110 Ala-scan analogs were tested in the same assay, 1 μM each.

In cultures, treatment of SH-SY5Y cells with P110 abolished MPP+-inducedproduction of mitochondrial superoxide, a major resource ofmitochondrial ROS (FIG. 6A and FIG. 7). The Alan-scan analogs of P110had limited or no activity relative to the effects of P110 onmitochondrial ROS production (FIG. 6A), suggesting that each of the 7amino acids of the fission inhibitor peptide sequence (SEQ ID NO:12) ofconstruct P110 contribute to the biological effects of the peptide.

Mitochondrial membrane potential was also determined using TMRM(tetramethylrhodamine methylester). Fifty μg of mitochondria wereincubated with recombinant Drp1 (50 ng) followed by incubation with TMRM(0.5 μM) in the presence or absence of P110 (1 μM) or NAC (2.5 mM).Fluorescence detection was performed using black 96-well plates in afluorescence microplate reader at 560 nm excitation and 690 nm emissionand potential was assessed by quenching of the fluorescent signal. Thedata are presented as mean±S.E. of percentage relative to controlmitochondria from three independent experiments (FIG. 6B, #, p<0.05 vs.control cells; *, p<0.05; **, P<0.01 vs. MPP+- or CCCP-treated cells).Isolated mouse liver mitochondria (50 μg) were incubated with Drp1recombinant protein (50 ng) in the presence or absence of P110 (1 μM). Akinetic change of mitochondrial superoxide production was determined byusing MITOSOX™ (red mitochondrial superoxide indicator). The data arepresented as mean±S.E. of percentage relative to the value at basallevel from three independent experiments (FIG. 6C, #, p<0.05 vs. controlmitochondria; *, p<0.05 vs. mitochondria with the addition of Drp1).Mitochondrial membrane potential of isolated mouse liver mitochondriawas determined by TMRM, as described above, in the indicated groups. NAC(2.5 mM) was used as a positive control (FIG. 6D). Protein levels ofcytochrome c in the mitochondria were determined by western blotanalysis with anti-cytochrome c antibodies in the indicated groups(insert, FIG. 6D). Total ROS production was determined by stainingSH-SY5Y cells with CM2HDCFA (1 μM 30 min at 37° C.) in the indicatedgroups. The data are presented as mean±S.E. of percentage relative tocontrol group from three independent experiments (FIG. 6E, *, p<0.05 vs.either MPP+ or CCCP treated-group; #, p<0.05 vs. control group).

Treatment with P110 significantly recovered mitochondrial membranepotential (MMP) in the presence of MPP+ or CCCP (FIG. 6B) and improvedthe assembly of the mitochondrial electron transport chain (ETC) (FIG.8) in the cultured SH-SY5Y cells treated with MPP+.

The oxidative phosphorylation system in the mitochondria is responsiblefor generating ATP and consists of five major membrane proteincomplexes, the mitochondrial complexes I-V. MPP+ is a specificmitochondrial complex I inhibitor. P110 was tested for potential effectson MPP+-induced defects in mitochondrial complexes. As shown in FIG. 8,in cultured SH-SY5Y cells, MPP+ treatment disassembled complex I and IV,as evidenced by the reduction of NDUFB8 (component of complex 1) andMTCOI (component of complex IV). By contrast, treatment of P110 underthe same conditions abolished the reduction of these two proteins,suggesting that P110 treatment recovered MPP+-induced oxidativephosphorylation defect and mitochondrial integrity. The upper panel ofFIG. 8 shows the western blot. The lower panel is a histogram in whichthe quantitative data generated from the western blot are expressed asmean±S.E. of 3 independent experiments.

To determine the direct effects of activated Drp1 on the mitochondria,Drp1 was incubated with isolated mouse liver mitochondria. Using theapproach as in (Johnson-Cadwell et al., 2007, J Neurochem101:1619-1631), mitochondrial superoxide production was measuredfollowed by the addition of Drp1 recombinant protein in the presence orabsence of P110

Interestingly, Drp1 addition triggered about a 50% increase inmitochondrial superoxide production over 8 minutes (p<0.05, n=3)relative to basal condition (FIG. 6C), Importantly, adding either P110or the Drp1 dominant negative mutant, Drp1K38A, abolished Drp1-inducedROS elevation in the mitochondria (FIG. 6 c, p<0.05, n=3). These datasuggest that activated Drp1 directly caused mitochondrial ROSproduction.

Further, after 8 min of Drp1 incubation with the mitochondria, thelevels of cytochrome c in the mitochondria declined, an effect that wasinhibited in the presence of P110 (FIG. 6D-top panel (western blot)),indicating that P110 inhibited cytochrome c release from themitochondria under these condition. Cytochrome c release is one of thesigns of mitochondrial membrane potential (MMP) dissipation.

The mitochondrial membrane potential of isolated mouse livermitochondria was then measured in the presence or absence of P110. Drp1addition caused a significant reduction of MMP, and treatment with P110improved the MMP, similar to that of the Drp1 K38A-treated group (FIG.6D, bottom panel). As expected, the anti-oxidant N-Acetyl cysteine(NAC), also prevented the dissipated MMP to the same extent as that ofP110. Finally, treatment with P110 greatly reduced total ROS produced bythe mitochondrial stressors, MPP+ and CCCP (FIG. 6E). Taken together,these data demonstrate that inhibition of stressor-inducedhyper-activation of Drp1 by P110 reduced mitochondrial damages bysuppressing mitochondrial ROS, improving mitochondrial membranepotential and mitochondrial integrity.

Example 7 Effects on Programmed Cell Death and Mitochondrial Integrity

Previous studies demonstrated that impairment of mitochondrial fissionis closely linked with increased apoptosis and autophagic cell death inresponse to various stimuli through increasing mitochondrialdepolarization and ROS (Wikstrom et al., 2009, Int J Biochem Cell Biol41:1914-1927). Moreover, Drp1 hyperactivation on the mitochondria hasbeen recently demonstrated to participate in TNFα-induced necrotic celldeath (Wang et al., 2012, Cell 148:228-243). Thus, Drp1-dependentmitochondrial dysfunction may represent a convergent point of severalprogrammed cell death (PCD) pathways. Experiments were performed todetermine if P110 affects programmed cell death under stress conditionsby inhibiting aberrant mitochondrial fission.

SH-SY5Y neuronal cells were treated with P110 followed by the exposureto MPP+(2 mM for 1 hour). Active form of Bax (NT-Bax), cytochrome c andBcl-2 on the mitochondria were determined by western blot analysis withthe indicated antibodies. Shown are representative data of threeindependent experiments. Quantification of the data are provided asmean±SE of three independent experiments.

P110 treatment inhibited early stages of apoptosis as shown by thegreatly reduced accumulation of active Bax on the mitochondria, blockedthe release of cytochrome c from the mitochondria and improved decreasedBcl-2 levels on the mitochondria in cultured SH-SY5Y neuronal cellstreated with MPP+ (FIG. 9A). The number of apoptotic SH-SY5Y cellsexposed to MPP+ was also greatly reduced by P110 treatment (FIG. 9B).Apoptosis was determined by annexin V staining 8 hours after MPP+exposure. The number of apoptotic cells was expressed as mean±S.E. ofpercentage relative to total number of cells.

Autophagy is another consequence of excessive mitochondrial fission(Twig et al., 2008, EMBO J. 27:433-446; Wikstrom et al., 2009, Int JBiochem Cell Biol 41:1914-1927). Autophagy can be measured by theinduction of the autophagic marker LC3 (microtubule-associated protein1, light chain 3, also known as ATG8). Cultured SH-SY5Y cells werestained by anti-LC3 antibodies and nuclei were stained by Hoechstsstaining. A histogram (FIG. 9C) depicts the number of LC3-positivepunctate/per cell in the indicated groups. The data are presented as themean±S.E. of three independent experiments. Western blot analysis of LC3I/II with anti-LC3 antibodies is shown and is presented as a histogramwhere the data are expressed as mean±S.E. of three independentexperiments (FIG. 9D, left panel).

Consistent with previous studies (Zhu et al., 2007, Am J Pathol170:75-86), MPP+caused excessive autophagy, as evidenced by theinduction of autophagic marker LC3 (microtubule-associated protein 1,light chain 3, also known as ATG8). Treatment with P110 reduced thenumber of LC3-positive puncti in the cells and LC3 cleavage (LC3 I toLC3 II) (FIGS. 9C and 9D, right panel), suggesting an inhibition ofexcessive autophagy.

Cell viability of stressed SH-SY5Y cells was measured by MTT assay.SH-SY5Y cells were treated with MPP+ (2 mM for 24 hours) followingtreatment with TAT, P110 or P110 Ala-scan analogs (1 μM each) (FIG. 9E,#, p<0.05 vs. control group; *, p<0.05, **, p<0.01 vs. MPP+-treatedcells).

Reduction in the PCD by treatment with P110 was also associated withimproved cell viability in cultured SH-SY5Y cells in response to stress(FIG. 9E). Consistent with results described above, treatment with P113or the seven P110 Ala-scan analogs had no significant effect on cellviability under the same conditions (FIG. 9E). Together, the data areconsistent with previous studies showing that Drp1 hyperactivation playsan active role in different types of cell death. Importantly, theselective mitochondrial fission inhibitor, P110, rescued cells fromthese cell death pathways.

In another experiment, Human SH-SY5Y cells were treated with Drp1 P108,P109 or P110 or Fis1 P111, P112 or P113 (0.5 μM) for 15 min followed bytreatment with MPP+ (1 mM for 24 hrs). The cell viability was thendetermined by MTT test.

Treatment with P109, P110, and P112 reduced mitochondrial damage andneuronal cell death (p<0.05 or p<0.01, n=7-8) that were triggered byMPP+ (1 mM for 24 hours). These peptides had no effects on mitochondrialhealth in control cells (Provisional FIG. 9F; *, p<0.05; **, p<0.01 vs.control cells, n=7-8).

Example 8 Effects on Neurite Degeneration of in a Parkinsonism Model

Aberrant mitochondrial fission has been highlighted in a number ofneurodegenerative diseases, such as parkinsonism, indicating a potentialmechanism by which mitochondrial dysfunction contributes toneurodegenerative diseases (Reddy et al., 2011, Brain Res Rev67:103-118). MPP+ induces selective degeneration of dopaminergic neuronsin a chemical experimental model of Parkinsonism. We thereforedetermined the effects of P110 treatment on the viability of primarydopaminergic neuronal cells in response to MPP+ exposure.

Primary rat dopaminergic neurons (cultured for 6 days) were treatedwithout or with P110 (1 μM) followed by treatment with or without MPP+(1 μM). Two his following MPP+ treatment, the cells were stained withMITOSOX™ red (to measure mitochondrial superoxide production) andanti-TH antibody (a marker of dopaminergic neurons). Fifteen hrs afterMPP+ treatment, cells were stained with anti-TH antibody and anti-Tom20antibody (a marker of mitochondria).

Quantitative results are provided for mitochondrial superoxideproduction (FIG. 10A), mitochondrial fragmentation (FIG. 10B) andneurite loss (FIG. 100) (as mean±SE of three independent experiments.Consistent with the findings above, treatment with P110 reducedmitochondrial fragmentation and mitochondrial ROS production in primarydopaminergic neurons exposed to MPP+. Importantly, P110 treatmentreduced neurite loss of dopaminergic neurons, which were identified bytyrosine hydroxylase (TH), a marker of dopaminergic neurons (FIG. 10C).These data suggest that inhibition of Drp1-induced mitochondrialdysfunction by P110 decreased neuronal degeneration in a cell culturemodel of Parkinsonism.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A mitochondrial fission inhibitor peptide consisting of 7, 8 or 9 amino acids, wherein the inhibitor peptide comprises an amino acid sequence which is 7 amino acids in length and which comprises the amino acid sequence as set forth in SEQ ID NO:12 having one, two or three conservative amino acid substitutions.
 2. The inhibitor peptide of claim 1, wherein the one, two or three amino acid substitutions are selected from the following: the aspartic acid at position 1 of SEQ ID NO:12 is substituted by glutamic acid, the leucine at position 2 or 3 of SEQ ID NO:12 is substituted by isoleucine, methionine, valine, or phenylalanine, the arginine at position 5 of SEQ ID NO:12 is substituted by lysine, the glycine at position 6 of SEQ ID NO:12 is substituted by alanine, threonine or serine, and the serine at position 7 of SEQ ID NO:12 is substituted by glycine, alanine or serine.
 3. A mitochondrial fission inhibitor linear peptide construct comprising: a) the fission inhibitor peptide according to claim 1; and b) a carrier peptide selected from the group consisting of SEQ ID NOs:31-45.
 4. The inhibitor linear peptide construct of claim 1, comprising the linker, wherein the linker is positioned between the fission inhibitor peptide and the carrier peptide, wherein the linker is selected from the group consisting of G, GG, GGG and GGGG (SEQ ID NO:62).
 5. A method for inhibiting or reducing abnormal mitochondrial fission, comprising contacting a cell with a composition comprising the mitochondrial fission inhibitor peptide of claim 1, wherein the contacting reduces abnormal mitochondrial fission.
 6. The method of claim 5, wherein the contacting a cell comprises administering the composition to an animal.
 7. The method of claim 6, wherein administering the composition to the animal results in a decrease in tremor, bradykinesia, rigidity, and/or postural dysfunction.
 8. A method of treating a subject suffering from or diagnosed with a disease or disorder associated with abnormal mitochondrial fission, comprising administering to the subject having or diagnosed with the disease or disorder a therapeutically effective amount of a pharmaceutical composition comprising a mitochondrial fission inhibitor construct, wherein the mitochondrial fission inhibitor construct comprises the peptide of claim 1, and wherein the administering is effective to reduce at least one adverse symptom of the disease.
 9. The method of claim 8, wherein the disease or disorder is Parkinson's disease, Huntington's disease, Alzheimer's disease, ischemia, reperfusion injury, diabetes-induced neuropathy, heart failure, or heart disease.
 10. A recombinant nucleotide vector comprising a nucleic acid sequence which encodes a mitochondrial fission inhibitor, a detectable marker, and origin of replication, wherein the nucleic acid sequence encoding the mitochondrial fission inhibitor comprises a nucleotide encoding the mitochondrial fission inhibitor peptide of claim
 1. 11. The recombinant nucleotide vector of claim 10, wherein the fission inhibitor construct encodes the fission inhibitor linear peptide construct comprising the fission inhibitor peptide and a carrier peptide.
 12. A method for measuring Drp1 GTPase inhibitory activity of a test peptide comprising: a) mixing Drp1 with the test peptide to generate a test reaction mixture; b) mixing Drp1 with no peptide to generate a control reaction mixture; c) measuring the level of GTPase activity in each of the control reaction and test reaction mixtures; and d) comparing the level of GTPase activity in the control reaction mixture with the level of GTPase activity in the test reaction mixture, e) identifying the test peptide as a mitochondrial fission inhibitor peptide when the level of GTPase activity in the test reaction mixture is about 50% to 100%, 40% to 60% or about 20% to 50% the level of GTPase activity in the control reaction mixture.
 13. The method according to claim 12, further comprising f) mixing MFN1, OPA1 or Dynamin 1 with the test peptide to generate a specificity test reaction mixture, g) mixing MFN1, OPA1 or Dynamin 1 with no peptide to generate a specificity control reaction mixture, h) measuring the level of GTPase activity in each of the specificity test reaction and specificity control reaction mixtures, i) comparing the level of GTPase activity in the specificity control reaction mixture with the level of GTPase activity in the test reaction mixture, and identifying the test peptide as a Drp1-specific mitochondrial fission inhibitor peptide when the level of GTPase activity in the test reaction mixture is about 90% to 100% or more than 100% of the level of GTPase activity in the specificity test reaction mixture.
 14. A mitochondrial fission inhibitor peptide consisting of 10, 11 or 12 amino acids, wherein the inhibitor peptide has at least 90% amino acid sequence identity to SEQ ID NO:10, and wherein the inhibitor inhibits a guanosine triphosphate phosphatase (GTPase) activity of the Dynamin 1-like protein (Drp1) (SEQ ID NO:1).
 15. A mitochondrial fission inhibitor linear peptide construct comprising: a) the fission inhibitor peptide according to claim 1; and b) a carrier peptide selected from the group consisting of SEQ ID NOs:31-45; wherein the mitochondrial fission inhibitor linear peptide construct is no more than 62 amino acids in length.
 16. The inhibitor construct of claim 15, wherein the carrier peptide is SEQ ID NO:32 or SEQ ID NO:33.
 17. The inhibitor linear peptide construct of claim 15, further comprising the a linker, wherein the linker is positioned between the fission inhibitor peptide and the carrier peptide, wherein the linker is selected from the group consisting of G, GG, GGG and GGGG (SEQ ID NO:62).
 18. The inhibitor linear peptide construct of claim 19, wherein the fission inhibitor peptide consists of SEQ ID NO:10.
 19. The inhibitor linear peptide construct of claim 17, wherein the linker is positioned between the fission inhibitor peptide and the carrier peptide, and wherein the linker is selected from the group consisting of G, GG, GGG and GGGG (SEQ ID NO:62). 